Catalog2015 16 by AGTC Bioproducts Ltd

National Diagnostics

Life Sciences Catalog 2015-16

Electrophoresis Histology Liquid Scintillation Reactive Oxygen Assays Electro-Optical Solvents nationaldiagnostics.com (800) 526-3867

National Diagnostics To Contact Technical Services

We encourage you to call our technical service department with any questions about National Diagnostics products or any molecular biology application.

USA: 800-526-3867 e-mail:

info@nationaldiagnostics.com

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office@agtcbioproducts.com

To Contact Customer Service

Our customer service department can be reached by phone, fax, or e-mail.

United States

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Europe

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35 YEARS OF INNOVATION National Diagnostics is a principle world source for chemicals and related products for scientific research. We are particularly proud of the innovations we have made to improve laboratory safety and the environmental responsibility of research. The National Diagnostics label on any product is an assurance of the highest level of quality and that all raw materials and finished products adhere to the most stringently controlled specifications. We place an emphasis on minimizing hazards and incorporating environmentally friendly features wherever possible. In combination with our exceptional technical and customer service staff, these products allow our customers to pursue scientific research in the knowledge that they are using the best, most reliable and safest products available.

OUR SPECIALTIES We specialize and have thrived in five areas of life science research: • Electrophoresis • Histology • Liquid Scintillation Counting • Electro-Optical • Oxygen Assay

OUR BRANDS National Diagnostics’ most recognizable brands include: • ProtoGel - The orginial Laemmli solution for protein separation. • UreaGel - The number one DNA/RNA solution. • Histo-Clear - The very first xylene substitute. • Ecoscint - The original biodegradable liquid scintillation cocktail • Opti-Clear - Award-winning clearing solvent Our dedication to quality applies to these, and all National Diagnostics products. The most demanding standards in the industry apply to the more than 150 products we manufacture.

GUARANTEE National Diagnostics, Inc. guarantees its products to be of the finest quality at the time of shipment. All materials shipped are guaranteed to meet the specifications indicated. A full refund, replacement or credit will be issued for any product which does not perform as specified due to a manufacturing defect. The limit of all claims relating to National Diagnostics, Inc. products shall be the invoice price of the product.

Distributors Worldwide

Please see the back cover for information regarding our global net work of distributors.

Visit us on the web

www.nationaldiagnostics.com

Products Applications Products

Product Sections Each color coded product section organizes products within basic categories. Product entries are accompanied by extensive cross-referencing to theoretical and practical discussions in the application sections. A table of related products is also presented.

Product Names in Alphabetical Order within a Category

Contact Information

Application Sections In addition to detailing the use of National Diagnostics products in the laboratory, the application sections of this catalog contain a wealth of practical and theoretical information useful to the experienced researcher. Furthermore, there are discussions of fundamental principles intended as a primer for beginning students.

Hundreds of diagrams and illustrations

Reactive Oxygen t Assays

Information Tables Related Products Cross References

Theoretical and Practical Discussions

Protocols

Alphabetical Product Index and Product Number Index Found at the back of the catalog, these indices provide order numbers, page numbers, and other quick ordering information.

Liquid t Scintillation

Indices

Electro-Optical t Solvents

Applications

Ordering Information

Suggested Reading

Histology Products

Liquid Scintillation Products

Histological Clearing Agents

Tissue Preparation

100

Biodegradable Sci n t i l l at ion C ockt ail s

Safer Aldehyde Disposal

102

Traditional Scintillation Cocktails

131

Mounting Media

103

Scintillation Cocktails for HPLC Flow Detectors

134

Histological Stains

104

Sample Oxidation Solutions

137

Tissue/Gel/Filter Solubilization

138

Radiation Safety

140

Accessories for Scintillation Counting

142

Autoradiography Image Enhancement

144

Histology Applications Histology Fundamentals 106 Fixation 107 Aldehyde-Based Fixatives 107 Other Fixatives 108 Factors Affecting Fixation 108 Working Safely with Fixatives 108 Safe Disposal of Aldehyde Waste 108 Decalcification 109 Processing Fixed Tissue 109 Dehydration 109 Clearing 109 Embedding 110 Sectioning 110 Frozen Sections 110 Artifacts in Histologic Sections 111 Staining 111 The Chemistry of Dyes 111 Why dyes produce color 112 The Chemistry of Staining 112 Staining Procedures 112 Mounting 113 Advanced Histological Techniques 114 Immunohistochemistry 115 Antibody Binding 115 Detection systems 115 Electron Microscopy 116 Fixation 117 Processing 117 Sectioning 117 Staining 117

Useful Information for the Histology Laboratory

118

Suggested Reading in Histology

119

Oxygen Radical Chemical Assays

126

Scintillators 145

Liquid Scintillation Applications Fundamentals of Liquid Scintillation Counting 146 Radioactive Emissions 147 Characteristics of Useful Isotopes 147 The Use of Isotopes in Research 147 Measurement of Radiation and Isotope Quantitation 147 Ionization Detection 148 Scintillation Detection 148 Mechanism of Liquid Scintillation Counting 148 Liquid Scintillation Signal Interpretation 149 Patterns of Light Emission 149 Pulse Analysis 150 Counting Efficiency 150 Quenching 150 The Complete Scintillation Cocktail 151 Sample Capacity 152 Chemiluminescence and Static Electricity 152 Waste Disposal Issues 152 LSC Applications 153 Counting Discrete Samples 154 Sample Neutralization (Elimination of Chemiluminescence) 154 Decolorizing 154 TLC Plates 155 Counting Samples on Cellulose-Ester Filters 155 Counting Tissue Samples 155 Counting 14CO2 156 Samples in Polyacrylamide Gels 156 Flow Liquid Scintillation 156 Liquid Scintillation and Radiation Safety 157 T r oubl e s h oot i n g L i quid Scin t i l l at ion E xpe r i m ent s 158

Reactive Oxygen Assays

120

Useful Information for Liquid Scintillation Counting

159

Fundamentals of Oxygen Radical Chemistry

122

Suggested Reading in Liquid Scintillation Counting

159

Electro-Optical Solvents Opti-Clear Solvent Overview

160

Product Information

161

Product Index

164

Subject Index

168

Indices

Electrophoresis

Electrophoresis Products

Products

Electrophoresis Products GEL MATRICES

BUFFERS

VISUALIZATION

ACCESSORIES

REAGENTS

GEL MATRICES P R O T E I N S E P A R A T I O N .................................6 - 9 ProtoGel

pg 6

30% or 40% stabilized 37.5:1 solution of acrylamide/bisacrylamide - ideal for the separation of proteins and polypeptides. ProtoGel Quick-Cast

pg 7

Ready-to-use premixed system making it possible to cast an SDS-PAGE gel in twenty minutes. AccuGel 29:1

pg 8

Ready-to-use monomer solution for the preparation of electrophoresis gels for Protein separation. AcrylaGel and Bis-AcrylaGel

pg 9

AcrylaGel is a 30% stabilized, ready-to-use acrylamide solution. Bis-AcrylaGel is a ready-to-use, 2% solution of methylene bisacrylamide.

PAGE GELS FOR DNA and RNA.................10 - 13

FASTER, EASIER, AND SAFER

With National Diagnostics ready-to-use matrix solutions, the time-consuming process of weighing, mixing, and filtering different reagents to prepare stock solutions is eliminated. You can now prepare the same gels faster, easier, and safer.

UreaGel 6 and 8

pg 10

Simply mix 4 parts of bottle one with 1 part of bottle two to formulate 19:1 denaturing DNA gels of constant percentage. UreaGel Systems

pg 10 - 11

Three bottle systems to cast 19:1 or 29:1 denaturing gels from 4% up to 20% quickly and conveniently. SequaGel MD

pg 12

For the detection of minor mutational differences in SSCP analysis and Heteroduplex analysis.

STABLE AND RELIABLE

The National Diagnostics label on any product is an assurance of the highest level of quality and that all raw materials and finished products adhere to the most stringently controlled specifications. Proprietary stabilization and purification methods produce the most reliable gel solutions on the market.

SequaGel XR

pg 12

National Diagnosticsâ&#x20AC;&#x2122; proprietary extended read matrix. AccuGel 19:1 and AccuGel 29:1

pg 13

Ready-to-use monomer solutions for the preparation of electrophoresis gels for DNA or RNA. AcrylaGel and Bis-AcrylaGel

pg 13

AcrylaGel is a 30% stabilized, ready-to-use acrylamide solution. Bis-AcrylaGel is a ready-to-use, 2% solution of methylene bisacrylamide.

AGAROSE ...................................................14 - 15

REPRODUCIBLE RESULTS

National Diagnosticsâ&#x20AC;&#x2122; products are selected and prepared specifically for electrophoretic applications, assuring superior results and reproducibility. You can trust that your results will be consistent from one electrophoretic run to another.

AquaPor LE

pg 14

High quality, general purpose agarose ideal for most routine applications AquaPor LM

pg 14

Low melting temperature agarose combining excellent handling characteristics with scrupulous quality certifications. AquaPor 3:1

pg 15

A specialty agarose providing excellent resolution of small DNA fragments. AquaPor HR

pg 15

High resolving, high strength, low melting agarose providing for the analysis and recovery of closely spaced DNA. AquaPor ES

pg 15

An ultra high strength/low EEO agarose for the separation of megabase size DNA fragments.

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Products

MOPS-SDS Running Buffer

pg 16

MES-SDS Running Buffer

pg 16

Tris-Glycine-SDS PAGE Buffer(10X)

pg 16

ProtoGel Buffers

pg 16

5X Protein Loading Buffer

pg 16

Protein Loading Buffer Blue (2X)

pg 16

Tris-Tricine-SDS PAGE Buffer(10X)

pg 16

Tris-Glycine Electroblotting Buffer (10X)

pg 17

PBS (10X)-Phosphate Buffered Saline

pg 17

Tris Buffered Saline 10X (+/- TWEEN)

pg 17

STOCK SOLUTIONS..............................................17 EDTA, 0.5M Sterile

pg 17

Potassium Chloride, 1M Sterile

pg 17

Sodium Chloride, 0.9% or 1M Sterile

pg 17

Tris HCl 1M pH 7.2, 7.4 or 7.6

pg 17

DNA/RNA ELECTROPHORESIS...............................18

TAE Buffer (50X)

pg 18

TBE Buffer(10X or 5X)

pg 18

TTE Glycerol Tolerant Buffer (20X)

pg 18

Triple Dye Loading Buffer (6X)

pg 18

UreaGel Loading Buffer

pg 18

DNA/RNA BLOT TING...........................................18

TE Buffer (100X)

pg 18

Denaturation Solution

pg 18

Neutralization Solution

pg 18

SSC Buffer (20X)

pg 18

VISUALIZATION AND SAMPLE PREP

PREPARING PROTEIN SAMPLES FOR PAGE.............26 ProtoGel Sample Prep Kit

pg 26

ND Protein Precipitation Kit

pg 26

Protein Loading Buffer Blue (2X)

pg 26

V I S U A L I Z I N G D N A a n d R N A . . . . . . . . . . . . . 27 - 28 Nuclistain

pg 27

Positive stain for the detection of DNA and RNA in gels Autofluor

pg 28

High resolution autoradiographic image intensifier Bromophenol Blue

pg 28

Bromocresol Green

pg 28

Xylene Cyanole FF

pg 28

GEL ACCESSORIES G E L AC C E SS O R I E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Gel Dry Film

pg 29

GelDry Film is a specially designed drying film for polyacrylamide gels. Glass Bond

pg 29

Temporarily affixes the PAGE gel to one of the glass casting plates. Glass Free

pg 29

Coats glass casting plates for easy release of polyacrylamide gels. Ion Exchange Resin

pg 29

Mixture of anionic and cationic resins. Ideal for deionizing acrylamide solutions.

ULTRA-PURE REAGENTS ULTRA-PURE REAGENTS....................................30 - 31 Acrylamide - Ultra Pure

pg 30

Ammonium Persulfate - Ultra Pure

pg 30

Bis - Ultra Pure (N,N’ - methylene bisacrylamide)

pg 30

Boric Acid - Ultra Pure

pg 30

DATD - Ultra Pure(N,N’ - diallyltartardiamide)

pg 30

Dextran Sulfate - Ultra Pure

pg 30

Dithiothreitol (DTT) - Ultra Pure

pg 30

POWDERED STAINS..............................................21

EDTA - Ultra Pure(Disodium ethylenediamine - tetraacetate dihydrate)

pg 30

Amido Black

pg 21

Reagent Alcohol

pg 30

Bromophenol Blue

pg 21

Formamide - Ultra Pure

pg 30

Coomassie Blue R-250

pg 21

Glycerol - Ultra Pure

pg 31

Coomassie Blue G-250

pg 21

Glycine - Ultra Pure

pg 31

WESTERN BLOTTING............................................22

2-Mercaptoethanol - Ultra Pure

pg 31

ProtoGlow ECL

Riboflavin - Ultra Pure

pg 31

SDS - Ultra Pure (Sodium Dodecylsulfate)

pg 31

SDS Solution (20%)

pg 31

TEMED (redistilled) - (N,N,N’,N’ - tetramethylethylene diamine)

pg 31

BLOTTING BUFFERS..............................................24

Tricine - Ultra Pure (N-Tris(hydroxymethyl methylglycine))

pg 31

Tris Buffered Saline 10X (+/- TWEEN)

pg 24

Tris - Ultra Pure (Tris(hydroxymethyl) aminomethane)

pg 31

ProtoBlock System

pg 24

Tween-20 - Ultra Pure

pg 31

PBS (10X) Phosphate Buffered Saline

pg 24

Urea - Ultra Pure

pg 31

Tris-Glycine Electroblotting Buffer (10X)

pg 24

Water - DEPC treated, sterile

pg 31

VISUALIZING PROTEIN..............................................19 ProtoStain Blue

pg 19

Safer, more sensitive colloidal Coomassie stain ProtoBlue Safe

pg 20

Eco-safe colloidal Coomassie stain. Sterling Rapid Silver Stain

Products

PROTEIN ELECTROPHORESIS AND BLOTTING...........16

Electrophoresis

BUFFER SOLUTIONS

pg 20

Silver staining in a faster and more convenient format

pg 22

Horseradish Peroxidase visualization system ProtoBlot Rapid Western Transfer Buffer

pg 23

ProtoLift Western Stripping Buffer

pg 23

PROTEIN STANDARDS...........................................25 Insite Markers

pg 25

ProtoMetrics

pg 25

ProtoMarkers

pg 25

USA: 1-800-526-3867 EUROPE: 441 482 646022

Protein Gels

Electrophoresis

Electrophoresis Products - Gel Matrices for Protein Separation

Gel Matrices for Protein Electrophoresis ProtoGel

ÂŽ

l l l l l

37.5 : 1 Acrylamide to Bisacrylamide Stabilized Solution Available in Either 30% or 40% Concentration Optimized for SDS-PAGE (Laemmli gels) of Proteins Consistently Crystal Clear Gels, Zero Fluorescence Stabilized for Long Shelf Life

30% Acrylamide

0.8% Bisacrylamide No Acrylic Acid

Every lot of ProtoGel is HPLC certified.

ProtoGel forms an electrophoresis matrix that is ideal for the separation of proteins and polypeptides. Available in either 30% or 40% concentration, ProtoGel is a stabilized, ready-to-use acrylamide/methylene bisacrylamide solution (37.5:1 ratio), manufactured from the highest quality materials, from which virtually all impurities have been removed. ProtoGel has zero acrylic acid content, eliminating the fixed charges that cause band streaking. Additionally, oxidation products such as aldehydes have been removed by a selective adsorption process. With ProtoGel, you can trust that your results will be consistent one electrophoretic run to the next. Storage: ProtoGel is stable for 24 months when stored tightly capped in a dark area at room temperature (20oC).

APPLIC ATION S 2-D Electrophoresis.....................79

Gel preparation:

Native protein Gels.............76 Western blotting ......................... 91

Denaturing protein gels.............. 69 Peptide Mapping .......................72

Gradient gels.......................70 Protein Purification ..................... 73

Molecular wt. determination......68

ProtoGel Buffers

Product Name

Cat. No.

Size

ProtoGel 30% EC-890 450 ml 1 Liter (1-3) 1 Liter (4 +) ProtoGel 40% EC-891 450 ml 1 Liter (1-3) 1 Liter (4 +)

Traditional Laemmli Buffer System l 18 Megohm Water/0.2 Micron Filtration The use of National Diagnosticsâ&#x20AC;&#x2122; ProtoGel Resolving Buffer and ProtoGel Stacking Buffer will ensure the purity and performance of your Laemmli gels. ProtoGel Buffer forms gels of 0.375 M Tris-HCl and 0.1% SDS, pH 8.8. ProtoGel Stacking Buffer forms gels of 0.125 M Tris-HCl and 0.1% SDS, pH 6.8. Storage: ProtoGel Resolving Buffer and ProtoGel Stacking Buffer are stable for 24 months when stored tightly capped in a dark area at room temperature (20oC). Product Name

USA: 1-800-526-3867 EUROPE: 441 482 646022

Cat. No.

Size

ProtoGel Resolving Buffer (4X) [pg 18] EC-892

450 ml 1 Liter (1-3) 1 Liter (4 +)

ProtoGel Stacking Buffer (4X)

EC-893

200 ml

EC-870 Tris-Glycine-SDS Buffer (10X) [pg 18]

1 Liter 4 Liter (1-3) 4 Liter (4+)

[pg 18]

Electrophoresis Products - Gel Matrices for Protein Separation

APP L IC ATION S Gel preparation:

2-D electrophoresis ....................79

Native protein Gels............ 76 Western blotting ......................... 91

Denaturing protein gels.......69 Peptide Mapping .......................72

Gradient gels.......................70 Protein Purification ..................... 73

Molecular wt. determination......72

Ideal for Western Blotting kDa 75 50 35

Economical and Convenient One Bottle System for Casting SDS-PAGE Gels

Ready to Run in 25 Minutes!

Ideal for Western Blotting Applications

Precast Convenience at a Fraction of the Price

ProtoGel Quick-Cast cuts the time required to cast a protein electrophoresis gel by 75%, and the number of steps by 2/3. Simply measure out the amount needed, add initiators and cast the gel. No mixing, no multiple measurements; just initiate, cast, and be ready to run in 20 minutes. With the reduced casting time, western blots can be finished within a regular working day:

Casting (prep and polymerization) 30 minutes Electrophoresis 1 hr Transfer (including prep time) 1.5 hr Blocking and Probing: 3 hrs Washing 1.5 hrs Detection 30 min Total Time 8 hr

Method of Use: ProtoGel Quick-Cast contains the monomers and buffer components to produce a 12% gel. 1. Measure out the volume of ProtoGel Quick-Cast needed to fill the cassette - typically 10ml for one mini-gel, 15ml for two.

2. Add 100 microliters of fresh 10% APS and 10 microliters of TEMED per 10ml ProtoGel Quick-Cast Mix briefly and pour into the gel cassette.

3. Insert comb and allow to polymerize at room temperature for 20 minutes.

10 Western blot analysis on total rat lung extract using anti-Flotillin antibody at 1:5000 dilution. Different dilutions of rat lung extract were resolved on a ProtoGel Quick-Cast gel and transferred to PVDF membrane.

Protein Gels

速

Electrophoresis

ProtoGel Quick-Cast 12%

ProtoGel Quick-Cast gels are run in standard 1X Tris-Glycine SDS. Electrical parameters will vary from apparatus to apparatus, but typically gels are run at 170V for 60 minutes ProtoGel Quick-Cast gels require no special handling after the run. Simply stain according to standard protocols. Product Name

Cat. No.

ProtoGel Quick-Cast 12% EC-895

Size 100 ml (13 gels) 450 ml (60 gels)

ProtoGel Quick-Cast Loading Buffer (2X) Developed for ProtoGel Quick-Cast 12%. Optimized to provide sharper bands and improved resolution. The combination of ProtoGel Quick-Cast and Quick-Cast Loading Buffer offers the fastest, easiest way to run high quality gels.

ProtoGel Quick-Cast Loading Buffer (2X)

EC-910

5 x 1ml

USA: 1-800-526-3867 EUROPE: 441 482 646022

Protein Gels

Electrophoresis

Electrophoresis Products - Gel Matrices for Protein Separation

AccuGel 2 9 :1

l l

AccuGel 19:1 is also available. See page 15.

l l

30% or 40%, 29:1 Acrylamide:Bisacrylamide Solution Stabilized for a Two Year Shelf Life Consistently Crystal Clear Gels For SDS-PAGE and Native DNA Electrophoresis

AccuGel 29:1 is a ready-to-use monomer solution for the preparation of electrophoresis gels for SDS-PAGE of proteins and the nondenaturing electrophoresis of DNA. AccuGel 29:1 contains 29 grams of acrylamide per gram MBA. Available in 30% or 40% concentration, AccuGel 29:1 is especially useful for protein scientists interested in the separation of smaller proteins by SDS-PAGE. Storage: AccuGel 29:1 is stable for 24 months when stored tightly capped in a dark area at room temperature (20OC). Product Name

APPLIC ATION S 2-D Electrophoresis.....................79

Gel preparation:

Native protein Gels.............76 Western blotting ......................... 91

Denaturing protein gels...............69 Peptide Mapping .......................72

Gradient gels.......................70 Protein Purification ..................... 73

Cat. No.

Size

AccuGel 29:1 (30%) EC-851 450 ml 1 Liter (1-3) 1 Liter (4 +) AccuGel 29:1 (40%) EC-852 450 ml 1 Liter (1-3) 1 Liter (4 +)

Molecular wt. determination......68

29:1 versus 37.5 :1 Which Acrylamide to Bis ratio is better for protein electrophoresis?

Both 29:1 and 37.5:1 gels are used in protein electrophoresis. The more commonly used 37.5:1 ratio represents the formulation in the original denaturing SDS-PAGE system of Laemmli (Nature, 1970). Despite the primacy of 37.5:1 in the literature, the 29:1 ratio has developed its own following with many committed practitioners. National Diagnostics has carried out a study comparing the performance of gels cast with AccuGel 29:1 to similar gels cast with our 37.5:1 solution, ProtoGel (EC-890). In this study the 29:1 ratio provided very slightly improved resolution of small proteins (<20kD) and the 37.5:1 ratio provided very slightly improved resolution of larger proteins (>80kD). These differences were very small, and in our opinion, either ratio will work well for the vast majority of SDS-PAGE applications.

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Products - Gel Matrices for Protein Separation

l l

Protein Gels

AcrylaGel a nd B is -Acryla Ge l

Acrylamide and Bisacrylamide Stabilized Solutions 18 megOhm Deionization, 0.2 Micron Filtration Aldehyde Free and Acrylic Acid Free Stabilized for a Two Year Shelf Life

Electrophoresis

AcrylaGel is a ready-to-use, 30% acrylamide solution in distilled/deionized water. Because acrylamide in its crystalline form is subject to self-polymerization, the purity and consistency of polymerized AcrylaGel is superior to that obtainable with powdered acrylamide. AcrylaGel can be crosslinked with Bis-AcrylaGel, a ready-to-use, 2% solution of methylene bisacrylamide with the same advantages as the AcrylaGel acrylamide solution. However, any powdered crosslinking reagent can also be used with AcrylaGel. Storage: AcrylaGel and Bis-AcrylaGel are stable for 24 months when stored tightly capped in a dark area at room temperature (20oC).

APP L IC ATIO N S Gel preparation:

Uracil interference ..................... 55

Denaturing DNA gels..........45 Methylation interference ........... 55

Native DNA gels ................56

Native protein Gels............ 76

Denaturing protein gels...... 69

Product Name

Cat. No.

Size

AcrylaGel EC-810 450 ml 1 Liter (1-3) 1 Liter (4 +) Bis-AcrylaGel EC-820 450 ml 1 Liter (1-3) 1 Liter (4 +)

Safe Sensitive Protein Visualization ProtoStain Blue

See page 21 for more information.

Eco-F riendly C olloid a l C oom a ssi e S t ai n ProtoStain Blue Colloidal Coomassie Blue G-250 stain is a premixed nonhazardous solution specially formulated for rapid, sensitive detection of proteins and safe, nonhazardous disposal. ProtoStain Blue is the most sensitive Colloidal Coomassie stain on the market, with the ability to detect less than 1ng of BSA. ProtoStain Blue contains no methanol, acetic acid, phosphoric acid or other hazardous components. Product Name

Cat. No.

ProtoStain Blue EC-727

Size 1 Liter

USA: 1-800-526-3867 EUROPE: 441 482 646022

DNA/RNA Gels

Electrophoresis

Electrophoresis Products - Gel Matrices for DNA & RNA Separation

Gel Matrices for DNA/RNA Electrophoresis SequaGel

UreaGel 29:1 Denaturing Gel System TM

l l l l

New!

The UreaGel 29:1 System allows researchers to easily and safely produce denaturing gels of the popular 29:1 acrylamide:bis-acrylamide formula in concentrations up to 20% monomer. These gels are ideal for analysis of RNA or DNA. The UreaGel 29:1 System consists of UreaGel 29:1 Concentrate, UreaGel Buffer and UreaGel Diluent. UreaGel 29:1 Concentrate contains 241.7g acrylamide, 8.3g methylene bisacrylamide per liter in 7.5M urea in a deionized aqueous solution. UreaGel Diluent consists of a deionized aqueous solution of 7.5M urea. UreaGel Buffer contains 0.89M Tris-Borate, 20mM EDTA and 7.5M urea at pH 8.3.

APPL IC A TION S Denaturing PAGE -

DNase I footprinting ................. 53

DNA/RNA45 ��45

RNA mapping ............................ 51

Sequencing:

S-1 mapping........................ 51

Sanger method .................. 48

RNAse protection................52

Maxam & Gilbert .............. 47

Uracil interference ..............55

Automated sequencers .......49

Methylation

Differential display .....................50

SequaGel

Casts 29:1 Denaturing Gels for RNA or DNA analysis Easily Cast up to 20% Gels Certified Nuclease Free Consistently Crystal Clear Gels

interference......................... 55

Storage: UreaGel 29:1 Concentrate, Diluent and Buffer are stable for one year when stored tightly capped in a dark area at room temperature. Product Name

Cat. No.

Size

UreaGel 29:1 System EC-829 1 Liter Kit 2.2 Liter Kit (1-3) 2.2 Liter Kit (4 +) UreaGel 29:1 Concentrate EC-828 450 ml 1 Liter (1-3) 1 Liter (4 +)

® ®

UreaGel 6 and 8 TM

l l l l

Ready-To-Use 19:1 Denaturing DNA Gel Solutions Certified Nuclease Free Consistently Crystal Clear Gels Twelve Month Shelf Life at Room Temperature

UreaGels 6 and 8 each consist of UreaGel Monomer Solution and UreaGel Complete Buffer Solution. The UreaGel Monomer Solution contains urea, as well as acrylamide and bis-acrylamide in the standard 19:1 ratio. UreaGel Complete Buffer Solution contains TBE and TEMED. Upon combining these two solutions, the researcher adds ammonium persulfate to form a crystal clear electrophoresis matrix with corresponding percent acrylamide (6 or 8) containing 1X TBE (89 mM Tris Base, 89 mM Boric Acid, 2 mM EDTA, pH 8.3) and 6M Urea. Easy to use and reliable, UreaGel 6 and 8 are well-regarded favorites of molecular biologists the world over. Product Name

Cat. No.

Size

UreaGel-6 EC-836 450 ml 1 Liter (1-3) 1 Liter (4 +) UreaGel-8 EC-838 450 ml 1 Liter (1-3) 1 Liter (4 +)

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Products - Gel Matrices for DNA & RNA Separation

UreaGel Sequencing System

Casts 19:1 Denaturing Gels up to 20% Monomer DNase & RNase Free Consistently Crystal Clear Gels Twelve Month Shelf Life at Room Temperature

l l l l

DNA/RNA Gels

Electrophoresis

SequaGel

The UreaGel Sequencing System consists of UreaGel Concentrate, UreaGel Diluent, and UreaGel Buffer. This system provides a convenient, dependable means for researchers to prepare gels of varying percentage. With the UreaGel Sequencing System the researcher readily prepares any commonly used gel formulation up to 20% monomer (19:1 acrylamide/bisacrylamide). Liter bottles of UreaGel Concentrate contain 237.5 grams of acrylamide, 12.5 grams of methylene bisacrylamide, and 7.5M urea in a deionized aqueous solution. UreaGel Diluent is supplied in 450 ml and 1 liter bottles containing 7.5M urea in deionized water. UreaGel Buffer is supplied in 100 ml and 200 ml bottles containing 0.89M Tris-Borate-20mM EDTA buffer pH 8.3 (10X TBE) and 7.5M urea.

AP P L IC A TION S Denaturing PAGE -

DNase I footprinting ................. 53

DNA/RNA45 ��45

RNA mapping ............................ 51

Sequencing:

S-1 mapping........................ 51

Sanger method .................. 48

RNAse protection................52

Maxam & Gilbert .............. 47

Uracil interference ..............55

Automated sequencers .......49

Differential display .....................50

Methylation

interference......................... 55

Storage: UreaGel Concentrate, UreaGel Diluent, and UreaGel Buffer are stable for one year when stored tightly capped, in a dark area at room temperature (20°C).

Product Name

Cat. No.

UreaGel Sequencing System*

EC-833

Includes UreaGel Diluent, Concentrate, and Buffer

Size 1 Liter Kit 2.2 Liter Kits (1-3) 2.2 Liter Kits (4 +)

UreaGel Diluent EC-840 450 ml 1 Liter (1-3) 1 Liter (4 +) UreaGel Concentrate EC-830 450 ml 1 Liter (1-3) 1 Liter (4 +)

Online: www.nationaldiagnostics.com

UreaGel Buffer EC-835 100 ml 200 ml (1-3) 200 ml (4 +) Tank B u f f e r TBE 10X [pg 20] EC-860 1 Liter 4 Liters (1-3) 4 Liters (4 +) U l t r a -P u r e I n iti a t o r s Ammonium Persulfate [pg 32] EC-504 25 grams 100 grams TEMED [pg 33] EC-503 25 ml

USA: 1-800-526-3867 EUROPE: 441 482 646022

DNA/RNA Gels

Electrophoresis

Electrophoresis Products - Gel Matrices for DNA & RNA Separation ®

SequaGel MD

l l l l

For the Detection of Minor Mutational Differences Point Mutation Analysis SSCP Analysis Heteroduplex Analysis

SequaGel MD permits minor mutational differences in DNA sequences to be detected as a high resolution relative mobility (Rf) shift. SequaGel MD is a proprietary formulation, supplied as a 2X stock, designed to resolve sequence related differences by SSCP (Single Strand Conformational Polymorphism) and heteroduplex analysis. For a detailed discussion of these techniques please see page 60 in the Electrophoresis Applications section of this catalog. Product Name

APPL IC A TION S Heteroduplex analysis ...............60 SSCP analysis............................. 61

Cat. No.

Size

SequaGel MD Monomer Solution EC-845 200 ml (1-3) 200 ml (4+) SequaGel MD Heteroduplex Kit EC-847 SequaGel MD Monomer Solution (200ml) Triple Dye Loading Buffer (1.2ml)

1 Kit

SequaGel MD SSCP Kit EC-846 1 Kit SequaGel MD Monomer Solution (200ml) SSCP Stop Solution (1.2ml)

SequaGel XR ®

Triple Dye Loading Buffer (6X) EC-855

1.2 ml

SequaGel MD SSCP Stop Solution EC-848

1.2 ml

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APPLIC ATIO N S

National Diagnostics’ SequaGel XR is specially formulated to produce greater resolution and longer read lengths. SequaGel XR’s optimized, proprietary formulation p revents aberrant band inversions and yields sharp, clear, highly resolved bands. SequaGel XR provides more information by allowing greater separation between bands near the top of the gel. This effect, similar to a wedge gel, is achieved with standard spacers. SequaGel XR is shipped as a two component system consisting of the SequaGel XR Monomer Solution and UreaGel Complete Buffer. UreaGel Complete Buffer Solution contains TBE and TEMED. No dilutions are necessary in this easy to use system. The researcher simply combines the two solutions, adds ammonium persulfate, and casts the gel. SequaGel XR is also available in the form of a 50% stock solution, SequaGel XR Concentrate.

Denaturing PAGE -

DNase I footprinting ................. 53

DNA/RNA45 ��45

RNA mapping ............................ 51

Product Name

Sequencing:

S-1 mapping........................ 51

Sanger method .................. 48

RNAse protection................52

Maxam & Gilbert .............. 47

Uracil interference ..............55

SequaGel XR EC-842 450 ml 1 Liter (1-3) 1 Liter (4 +)

Automated sequencers .......49

Methylation

Differential display .....................50

Extended Range Gel Solution for DNA Electrophoresis Higher Resolution Ideal for LICOR Sequencers Available as a Premixed 2 Bottle Kit or Concentrate

interference......................... 55

USA: 1-800-526-3867 EUROPE: 441 482 646022

Cat. No.

Size

SequaGel XR Concentrate EC-843 100 ml 450 ml (1-3) 450 ml (4 +)

Electrophoresis Products - Gel Matrices for DNA & RNA Separation

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Premixed Acrylamide:Bisacrylamide Solutions Stabilized for a Two Year Shelf Life Consistently Crystal Clear Gels Certified Nuclease Free

DNA/RNA Gels

Electrophoresis

AccuGel 19:1 and AccuGel 29:1

AccuGel 19:1 and AccuGel 29:1 are ready-to-use monomer solutions for the preparation of electrophoresis gels for DNA or RNA. AccuGel 19:1 contains 19 grams of acrylamide per gram of methylene bisacrylamide (MBA) cross-linker; AccuGel 29:1 contains 29 grams of acrylamide per gram MBA. AccuGel 19:1 ratio is intended for the denaturing electrophoresis of DNA and AccuGel 29:1 is intended for native DNA electrophoresis and SDS-PAGE of proteins.

APP L IC ATIO N S Nondenaturing PAGE-DNA.......56 Ribonuclease protection ........... 52 Denaturing PAGE-DNA/RNA.....45 Uracil interference ..................... 55 Sequencing:

Gel preparation:

Denaturing DNA gels..........45

Sanger method .................. 48

Native DNA gels ................56

Maxam & Gilbert .............. 47

Automated sequencers .......49

Gel electrophoresis of

Storage: AccuGel 19:1 and AccuGel 29:1 are stable for 24 months when stored tightly capped in a dark area at room temperature (20oC). Product Name

Cat. No.

Size

AccuGel 19:1 (30%) EC-849 450 ml 1 Liter (1-3) 1 Liter (4 +) AccuGel 19:1 (40%) EC-850 450 ml 1 Liter (1-3) 1 Liter (4 +)

PCR products ..............................58

AccuGel 29:1 is also suitable for protein applications. See page 10.

AccuGel 29:1 (30%) EC-851 450 ml 1 Liter (1-3) 1 Liter (4 +) AccuGel 29:1 (40%) EC-852 450 ml 1 Liter (1-3) 1 Liter (4 +)

AcrylaGel and Bis-AcrylaGel TM

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Acrylamide and Bisacrylamide Stabilized Solutions 18 megOhm Deionization, 0.2 Micron Filtration Certified Nuclease Free Stabilized for a Two Year Shelf Life

AcrylaGel is a ready-to-use, 30% acrylamide solution in distilled/deionized water. AcrylaGel can be crosslinked with Bis-AcrylaGel, a ready-to-use, 2% solution of methylene bisacrylamide with the same advantages as the AcrylaGel acrylamide solution. However, any powdered crosslinking reagent can also be used with AcrylaGel. Storage: AcrylaGel and Bis-AcrylaGel are stable for 24 months when stored tightly capped in a dark area at room temperature (20oC). Product Name

APP L IC ATIO N S Gel preparation:

Uracil interference ..................... 55

Denaturing DNA gels..........45 Methylation interference ........... 55

Native DNA gels ................56

Native protein Gels............ 76

Denaturing protein gels...... 69

Cat. No.

Size

AcrylaGel EC-810 450 ml 1 Liter (1-3) 1 Liter (4 +) Bis-AcrylaGel EC-820 450 ml 1 Liter (1-3) 1 Liter (4 +)

USA: 1-800-526-3867 EUROPE: 441 482 646022

Agarose Matrices

Electrophoresis

Electrophoresis Products - Gel Matrices - Agarose

AquaPor GTAC Agarose TM

AquaPor LE EEO (-mr)

<0.12

AquaPor LM

AquaPor HR

<0.05

<0.12

AquaPor ES

AquaPor 3:1

<0.05

>1200 @ 1.0% >450 @ 1.0% >400 @ 1.5% Gel Strength >1100 @ 3.0% (g/cm2)

>1700 @ 1.0% >3200 @ 1.5%

<36 @ 1.0% <27 @ 1.5% <36 @ 1.5% <40 @ 1.5% Gel Temp (oC)

<0.07 >1200 @ 1.0% >6500 @ 4.0% <40 @ 4%

<89 @ 1.0% <65 @ 1.5% <80 @ 1.5% <89 @ 1.5% <92@ 4% Melt Temp (oC) Sulfate (%)

<0.20

DNase,RNase, None Protease

<0.20

None

National Diagnostics AquaPor Agaroses are extremely reliable and convenient to work with. They dissolve easily with low boil over to produce clear, strong gels. National Diagnosticsâ&#x20AC;&#x2122; agarose is the most rigorously tested in the industry. Each AquaPor scrupulously adheres to stringently controlled specifications. All AquaPor agaroses are Genetic Technology Analysis Certified (GTAC). Every procedure for which an AquaPor is intended has been tried and tested, and every AquaPor Agarose is certified DNase, RNase, and protease free.

AquaPor LE GTAC l l

APPLIC A TION S Agarose gel electrophoresis.......62 Restriction digest mapping..........63 Preparing agarose gels.............. 62 Agarose electrophoresis-RNA......... Southern blotting.........................84 67 Northern blotting .......................83 Immuno-Electrophoresis............. 78 Purifying DNA/RNA-Agarose ....... Radial Immuno-Diffusion ........... 78 64

AquaPor LM GTAC

AquaPor LE GTAC is a high quality, general purpose agarose gel material ideal for most routine applications. Low EEO reduces diffusion of smaller fragments and results in sharper, more clearly defined bands. High gel strength facilitates ease of use and handling of gels. AquaPor LE is certified to contain no detectable DNase, RNase, or protease. It makes an excellent matrix for resolution of nucleic acids as well as high molecular weight proteins. AquaPor LE may be used confidently for Southern, Northern, and Western blotting, as well as in-gel hybridizations. Product Name

APPLIC A TION S Preparing agarose gels.............. 62 In gel restriction digestion ..........65 Purifying DNA/RNA................. -64 Restriction digest mapping..........63 In gel ligation...............................65

Cat. No.

Size

AquaPor LE GTAC EC-202 25 g 100 g (1 - 3) 100 g (4 +) 500 g

l l

Agarose gel electrophoresis.......62 In gel PCR amplification..............65

Molecular Biology Grade Agarose Low Electroendosmosis

Easy-to-Handle Low Melt Agarose Analysis/Recovery of Large and Small Fragments

AquaPor LM GTAC is a low melting temperature agarose for both large and small DNA fragments. For large fragments up to 25 kb, a 1% AquaPor LM solution forms a gel strong enough to be handled without fracturing. For separation of smaller fragments from 20 bp to 1000 bp, AquaPor LM possesses low viscosity so that 3% or 4% gels can be made. DNA fragments separated in AquaPor LM may be immediately used for enzymatic manipulation in the presence of remelted agarose. Extra advantages: AquaPor LM is GTAC certified to be DNase, RNase, and protease free. AquaPor LM is certified for in-gel PCR (re)amplification and in-gel ligation, and will not affect transformation efficiencies. Product Name

Cat. No.

Size

AquaPor LM GTAC EC-204 25 g 100 g (1 - 3) 100 g (4 +)

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Products - Gel Matrices - Agarose

APP L IC ATIO N S Agarose gel electrophoresis.......62 Restriction digest mapping..........63 Preparing agarose gels.............. 62 Agarose electrophoresis - RNA...... 67

AquaPor HR GTAC

APP L IC ATIO N S Preparing agarose gels.............. 62 Agarose electrophoresis-RNA......... Purifying DNA/RNA-Agarose ....... 67 DNase I footprinting ..................53

64 Gel electrophoresis of

Small Fragment DNA and RNA RT-PCR

AquaPor 3:1 GTAC is a molecular biology grade agarose specifically manufactured to yield strong gels for fine resolution of small DNA. It makes an excellent matrix for resolution of small fragment DNA and RNA. AquaPor 3:1 may be used confidently for Southern and Northern blotting, as well as DNase footprinting and RT-PCR. AquaPor 3:1 is GTAC certified to be DNase, RNase, and protease free. Product Name

Cat. No.

Size

AquaPor 3:1 GTAC EC-206

25 g 100 g (1 - 3) 100 g (4 +)

l l

Agarose gel electrophoresis.......62 Restriction digest mapping..........63

Agarose Matrices

Electrophoresis

AquaPor 3:1 GTAC

High Strength, High Resolution Agarose Analysis/Recovery of Small DNA

AquaPor HR GTAC is our highest resolving agarose, specifically manufactured for optimal electrophoretic separation of small DNA fragments, PCR products, and proteins. AquaPor HR will resolve DNA down to 2% size difference. Uniform particle size minimizes boil over. Additionally, the high quality of this agarose has allowed the certification of AquaPor HR for PCR (re)amplification directly in the presence of the remelted gel. AquaPor HR is GTAC certified to be DNase, RNase, and protease free, and certified for in-gel PCR. Product Name

Cat. No.

Size

AquaPor HR GTAC EC-205 25 g 100 g (1 - 3) 100 g (4 +)

PCR Products ..............................57

AquaPor ES GTAC l l

APP L IC ATIO N S Agarose gel electrophoresis.......62 PFGE/FIGE .................................66 Preparing agarose gels.............. 62

Ultra High Strength/Low EEO Agarose Blotting of MegaBase DNA

The very low EEO of AquaPor ES will significantly reduce electrophoresis times for Pulsed Field Gel (PFG) applications, and also increase band sharpness. The extremely high gel strength makes it possible to cast gels as low as 0.3% which remain intact through staining, destaining, photographic documentation, and blotting. These low percentage gels may be used for the electrophoretic separation of DNA fragments as large as 50 kb, and macromolecular protein complexes from 500 kDa to 10 MDa size. Product Name

Cat. No.

Size

AquaPor ES GTAC EC-203 25 g 100 g (1 - 3) 100 g (4 +) USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis

Proteomics Buffers

Electrophoresis Products - Buffers for Proteomics

Buffer Solutions The quality of the buffers determines the electrical parameters of electrophoresis and the structure and solubility of the biomolecules under study. Buffer quality is as important for electrophoresis results as the gel matrix itself. Produced to the most exacting standards in the industry, National Diagnostics’ buffers are stable and pure. They eliminate weighing and mixing, save time, and help ensure reproducible results. l

Stringent Quality Control l Save Money

l 18

Megohm Water l Save Time

l 0.2

Micron Filtration l Improve Results

P R OTEI N E LE CTROPHORE SI S BUFFE R S

MOPS-SDS Running Buffer (20X)

Tris-Tricine-SDS PAGE Buffer (10X)

A concentrated solution of Tris-Tricine-SDS. Intended for use as the running buffer Ideal running buffer for high resolution SDS-PAGE applications. When diluted, the in SDS polyacrylamide gel electrophoresis (PAGE) for resolving smaller proteins ( buffer contains 50mM Tris, 50mM MOPS, 0.1% SDS and 1mM EDTA. 5kD to 20kD) that cannot be resolved with the traditional Laemmli buffer system. MOPS-SDS Running Buffer (20X) EC-867

450 ml 1 Liter

MES-SDS Running Buffer (20X) Popular buffer formula using the highest quality components and processed through rigorous production and quality control standards. When diluted, the buffer contains 50mM Tris, 50mM MES, 0.1% SDS and 1mM EDTA. MES-SDS Running Buffer (20X)

EC-868

450 ml 1 Liter

Tris-Glycine-SDS PAGE Buffer (10X) Tris-Glycine-SDS-PAGE Buffer (10X) is a concentrated solution for use as the running buffer in SDS-PAGE of proteins. Each bottle contains 0.25M Tris base, 1.92M glycine, and 1% (w/v) SDS. Tris-Glycine-SDS PAGE Buffer (10X) EC-870 1 Liter 4 Liters (1-3) 4 Liters (4 +)

M

ProtoGel Buffers National Diagnostics’ ProtoGel Resolving Buffer and ProtoGel Stacking Buffer will ensure the purity and performance of your Laemmli gels. ProtoGel Resolving Buffer forms gels of .375 M Tris-HCl and 0.1% SDS, pH 8.8. ProtoGel Stacking Buffer forms gels of .125 M Tris-HCl and 0.1% SDS, pH 6.8. 4X ProtoGel Resolving Buffer EC-892 450 ml 1 Liter (1-3) 1 Liter (4 +) 4X ProtoGel Stacking Buffer EC-893 200 ml

Tris-Tricine-SDS PAGE Buffer (10X) EC-869

1 Liter

5X Protein Loading Buffer 5X Protein Loading Buffer is a reducing sample buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 5X Protein Loading Buffer contains Tris-HCl (pH 8.5), Lithium Dodecyl Sulfate, 50% glycerol, EDTA, DTT and tracking dye in distilled/deionized water.

5X Protein Loading Buffer

EC-887

10 x 1ml

Protein Loading Buffer Blue (2X) Protein Loading Buffer Blue (2X) is a reducing sample preparation buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Loading Buffer Blue (2X) contains bromophenol blue as a tracking dye, in solution with 0.5M Tris-HCl (pH 6.8), 4.4% (w/v) SDS, 20% (v/v) glycerol, 2% (v/v) 2-mercaptoethanol, in distilled/deionized water. Protein Loading Buffer Blue (2X) EC-886

10 x 1ml

ProtoGel Quick-Cast Loading Buffer (2X) Recommended for use with ProtoGel Quick-Cast 12%. Provides sharpest bands and improved resolution. ProtoGel Quick-Cast Loading Buffer (2X)EC-896 5 x 1ml

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Products - Buffers for Genomics

TBS (10X) Tris Buffered Saline

0.5M solution. Autoclaved.

EDTA

Ultra-pure, clear, autoclaved RNase free solution. After dilution to 1X, TBS has the following concentrations: 25 mM Tris; 138 mM NaCl and 2.7mM potassium chloride. TBS (10X)

EC-881

1 Liter

EDTA EC-900 1 Liter

Potassium Chloride 1M solution. Autoclaved. Potassium Chloride

TBST (10X) Tris Buffered Saline w/ Tween-20

Genomics Buffers

STOCK BUFFER SOLUTIONS

Electrophoresis

PROTEIN BLOTTING BUFFERS

EC-903

1 Liter

Sodium Chloride Formulated in both 1M and 0.9 percent editions. Autoclaved.

Same outstanding quality as our TBS buffer with added Tween-20 component. When diluted to 1X, TBST has the following concentrations: 25 mM Tris; 138 mM NaCl, 2.7mM potassium chloride and 0.05% Tween-20. TBST (10X)

EC-882

Sodium Chloride

0.9% 1M

EC-901 EC-902

1 Liter 1 Liter

1 Liter

Tris-HCl PBS (10X) Phosphate Buffered Saline Produced as a 10X concentrate, National Diagnosticsâ&#x20AC;&#x2122; PBS is a clear, RNase free solution. After dilution to 1X, PBS has the following electrolyte concentrations: 137 mM NaCl; 2.7 mM KCl; 10mM Phosphate Buffer. PBS (10X) CL-253 450 ml 1 Liter 4 Liters

Tris-Glycine Electroblotting Buffer (10X) Tris-Glycine Electroblotting Buffer (10X) is a concentrated solution of the standard tank buffer for Western electroblotting procedures. To prepare 1 liter of working strength buffer, add 100 ml of Tris-Glycine Electroblotting Buffer to 200 ml of methanol and 700 ml of distilled/deionized water. Tris-Glycine Electroblotting Buffer (10X) contains 0.25M Tris base and 1.92M glycine. Tris-Glycine Electroblotting Buffer(10X) EC-880

1 Liter 4 Liters (1-3) 4 Liters (4 +)

Popular formulation of Tris hydrochloride. Available in 1M solutions at pH 7.2, 7.4 and 7.6. Tris-HCl

pH 7.2 pH 7.4 pH 7.6

EC-922 EC-923 EC-925

1 Liter 1 Liter 1 Liter

Sodium Acetate 3M solutions optimized for genomic precipitation. Autoclaved and ultrapure. Available in pH 4.5, 5.2, and 7.0. Sodium Acetate

pH 4.5 pH 5.2 pH 7.0

EC-905 EC-906 EC-907

200 ml 200 ml 200 ml

Potassium Acetate Ultrapure, autoclaved solutions. Available in two concentrations. Potassium Acetate

1M 5M

EC-908 EC-909

1 Liter 1 Liter

Online: www.nationaldiagnostics.com

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis

Protein Visualization

Electrophoresis Products - Protein Visualization

DNA/RNA ELECTROPHORESIS MESA RNA Electrophoresis Buffer

TE Buffer (100X)

An RNase-free buffer (MOPS/EDTA/Sodium acetate) for RNA electrophoresis. Used as both the tank and gel buffer for denaturing RNA agarose electrophoresis .

TE Buffer is an autoclaved 100X concentrated solution of 1M Tris-HCl, pH 8, with 0.1 M Na2EDTA. This buffer is the standard for DNA and RNA purification, processing and storage. National Diagnostics’ TE buffer is produced to the same exacting standards that have made all our buffers trusted by molecular biologists worldwide.

MESA RNA Electrophoresis Buffer EC-911

TE Buffer 100X

TAE Buffer (50X) TAE Buffer (50X) is a concentrated solution of 2M Tris-Acetate and 100mM Na2EDTA in distilled/deionized water (pH 8.3 at 1X concentration). TAE Buffer is used for agarose DNA electrophoresis. TAE Buffer 50X EC-872 1 Liter (1-3) 1 Liter (4 +)

TBE Buffer (10X or 5X) National Diagnostics’ TBE Buffer is a concentrated buffer solution of Tris-Borate-EDTA in distilled/deionized water. TBE is ready-to-use as a 10X or 5X concentrate. When diluted, the 1X solution contains 0.089M Tris base, 0.089M boric acid (pH 8.3) and 2mM Na2EDTA. TBE Buffer 10X EC-860 1 Liter 4 Liters (1-3) 4 Liters (4 +) TBE Buffer 5X EC-861 1 Liter 4 Liters (1-3) 4 Liters (4 +)

TTE Glycerol Tolerant Buffer (20X) National Diagnostics’ TTE Glycerol Tolerant Buffer (20X) is a concentrated Tris-Taurine-EDTA buffer solution in distilled/deionized water. TTE (20X) eliminates the band distortion associated with DNA samples in glycerol. National Diagnostics’ TTE Glycerol Tolerant Buffer (20X) is supplied in 1 liter bottles, containing 1.78M Tris base, 0.57M Taurine, and 10mM Na2EDTA. TTE Glycerol Tolerant Buffer (20X) EC-871 1 Liter (1-3) 1 Liter (4 +)

UreaGel Loading Buffer is a denaturing loading buffer for UreaGel and other denaturing polyacrylamide gel applications. UreaGel Loading Buffer contains 95% Formamide, 18mM EDTA, SDS, Xylene Cyanol and Bromophenol Blue. UreaGel Loading Buffer

EC-857

EC-862

25 ml

Denaturation Solution National Diagnostics’ Denaturation Solution is a ready-to-use buffer solution of sodium chloride and sodium hydroxide in distilled/deionized water. It is especially designed for use in Southern and Northern Blotting, and in situ hybridization procedures. Denaturation Solution contains 1.5M sodium chloride and 0.5M sodium hydroxide. Denaturation Solution EC-875 1 Liter (1-3) 1 Liter (4 +)

Neutralization Solution National Diagnostics’ Neutralization Solution is a ready-to-use sodium chloride-Tris buffer solution. It is especially designed for use in Southern and Northern Blotting, and in situ hybridization procedures. Neutralization Solution contains 3M sodium chloride and 0.5M Tris in distilled/deionized water. Neutralization Solution EC-876 1 Liter (1-3) 1 Liter (4 +)

SSC Buffer (20X) SSC Buffer (20X) is a concentrated solution of sodium chloride-sodium citrate in distilled/deionized water. SSC Buffer is widely used in nucleic acid blotting and hybridization protocols. SSC Concentrate (20X) is already in solution, which eliminates the weighing, mixing, and adjusting of pH necessary with powdered buffers. The 20X formulation allows for easy dilutions to all needed concentrations. SSC Buffer Concentrate (20X) contains 3M sodium chloride and 0.3M sodium citrate (pH 7.0). SSC Buffer (20X) EC-873 1 Liter 4 Liters

UreaGel Loading Buffer

10 x 1 ml

20X SSPE Washing buffer for Northern and Southern blotting. DNase and RNase free.

20X SSPE

EC-910

Triple Dye Loading Buffer (6X)

Denhardt’s Solution

With three tracking dyes (bromophenol blue, xylene cyanole, and Orange G), Triple Dye Loading Buffer (6X) is a nondenaturing loading buffer for native polyacrylamide and agarose gel applications. Triple Dye Loading Buffer contains 50% (w/v) sucrose and 40mM Tris base in distilled, deionized water.

Component of hybridization solutions for Northern and Southern blotting. Increases speed and sensitivity of hybridization reactions.

Triple Dye Loading Buffer 6X

DNA/RNA BLOTTING

EC-855

USA: 1-800-526-3867 EUROPE: 441 482 646022

1.2 ml

Denhardt’s Solution

EC-915

50 ml

Electrophoresis Products - Protein Visualization Electrophoresis

Protein Visualization

Electrophoresis Detection and Visualization Systems Whether you are working with stains, radioactivity, fluorescence, chemiluminescence or bioluminescence, our products can help improve your results. National Diagnostics offers a range of visualizing agents and enhancers to make detection more sensitive with increased efficiency.

ProtoStain Blue TM

• Fastest, Most Sensitive Coomassie Stain • Ready to Use: No Measuring or Mixing • Detects As Little As 1ng of BSA

ProtoStain Blue is a colloidal Coomassie Blue G-250 solution that is a safer, more sensitive method for detecting proteins on electrophoresis gels. Bands begin to appear in only 15 minutes, and ProtoStain Blue has the ability to detect down to 1ng denatured BSA.

Fast Protocol - No mixing or measuring Wash gel 3 times for 10 minutes with deionized water on an orbital shaker.

ProtoStain Blue is safe and nonhazardous, containing no methanol, acetic acid or phosphoric acid.

Nanogram Sensitivity

Add enough staining solution to completely cover the gel (20 - 50 ml). Bands containing more than 1µg of protein will be detected within 15 minutes. (For maximum sensitivity incubate the gel in stain for at least 4-5 hours.)

A P P L I CA T I O NS Staining proteins in gels..............86 Colloidal Coomassie...................87

12% ProtoGel, loaded with indicated dilutions of BSA and Ovalbumin, and stained with ProtoStain Blue.

Product Name

Cat. No.

Size

ProtoStain Blue EC-727 1 Liter USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis

Protein Visualization

Electrophoresis Products - Protein Visualization

ProtoBlue Safe TM

Eco-Friendly, Ultra Sensitive Colloidal Coomassie G-250 Stain 速

Compared to similar stains, the more finely controlled colloidal structure of ProtoBlue Safe improves both the sensitivity and the universality of staining. ProtoBlue Safe is less prone to high background caused by trace residual SDS in the gel.

Costs Less than Regular Coomassie Laboratories typically spend twice as much per gel in methanol and acetic acid for staining and destaining with regular Coomassie than for ProtoBlue Safe (including ethanol cost). In addition, ProtoBlue Safe is much faster, more sensitive, and can be poured down the drain after use.

Nonhazardous Disposal Used stain solution is not hazardous waste (as defined by United States Title 40 Code of Federal Regulations (40 CFR 261.24(a)). Sink disposal of ProtoBlue Safe is permitted in most locations. Long Shelf Life - ProtoBlue Safe is stable for two years stored at room temperature in a cool, dry place.

Product Name

Cat. No.

Size

ProtoBlue Safe EC-722 450 ml 1 Liter 4 Liter Coomassie速 is a registered trademark of Imperial Chemical Industries, PLC.

Sterling Rapid Silver Stain TM

l l l

Detects Sub-Nanogram Levels of DNA or 5ng of Protein Fix, Wash and Stain in One Hour Easy System with Fewer Reagents and One Stain

The Sterling Silver Staining System offers high sensitivity silver staining faster and more conveniently than any other kit. The unique chemistry of Sterling allows staining using only one solution. Simply fix a polyacrylamide gel using our unique fixative, wash, and place the gel in the staining solution. Bands will appear in 5-10 minutes. The Sterling Silver Staining System stains 18 mini-gels.

APPL IC ATIO N S Silver staining proteins................89 Silver staining DNA.....................82

USA: 1-800-526-3867 EUROPE: 441 482 646022

Product Name

Cat. No.

Size

Sterling Kit EC-720 1 kit (1-3) 1 kit (4+)

Electrophoresis Products - Protein Visualization Electrophoresis

Protein Visualization

Powdered Stains Amido Black

Amido Black was one of the first dyes used to detect proteins on electrophoretic gels. It is not as sensitive as Coomassie Blue R-250 for most proteins, but it can be a better stain for some acidic peptides which stain poorly in Coomassie. Product Name Amido Black 10B

Bromophenol Blue

Cat. No.

Size

HS-601 25 g

Bromophenol Blue is used as a tracking dye, because its charge/mass ratio allows it to comigrate with smaller macromolecules through PAGE and Agarose gels. The dye undergoes a color shift to yellow at acidic pH. Product Name

Cat. No.

Size

Bromophenol Blue HS-603 10 g

Coomassie Blue G-250 速

速

Coomassie Blue G-250 is a useful stain for protein detection in PAGE gels. Coomassie staining gives blue bands on a clear background, with a sensitivity of 100 - 500 ng/band. The G-250 dye is converted to a leuco form below pH 2-3. The leuco form regains color on protein binding, and is the basis for the Bradford Protein Assay. Product Name

Cat. No.

Size

Coomassie Blue G-250

HS-605

10 g

Coomassie Blue R-250 is a sensitive stain for protein detection in PAGE gels. Coomassie staining gives blue bands on a clear background, with a sensitivity of 50-100 ng/band. Product Name

Cat. No.

Size

Coomassie Blue R-250

HS-604

10 g

速

Coomassie is a registered trademark of Imperial Industries, PLC.

Coomassie Blue R-250 USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis

Protein Visualization

Electrophoresis Products - Protein Visualization

Western Blotting ProtoGlow ECL l l l

Extended, Long Lasting Signal Life Long Shelf Life Less Antibody Needed

National Diagnostics' ProtoGlow ECL delivers the latest technology developed for enhanced chemiluminescent detection on Western blots. The unique chemistry of the ProtoGlow ECL system increases the sensitivity of Western blots by up to 20 fold. This allows the detection of proteins at much lower abundance and/or the use of higher dilutions of primary and secondary antibodies, economizing on these expensive reagents.

Improved Sensitivity The improved sensitivity of ProtoGlow ECL is demonstrated below. Identical dot blots of transferrin were blocked and probed with the same reagents, and then subjected to detection using either ProtoGlow ECL or the most popular competitor. ProtoGlow ECL detected 20 fold less sample than the competitor's products.

Long Lasting Signal ProtoGlow ECL generates a consistent light output for as long as 120 minutes. This ensures results are more reproducible: exposure to exposure, blot to blot. It also allows multiple exposures of a single blot to be taken to optimize the signal:noise ratio.

Nitrocellulose dot blot probed with HRP-labelled antibody at 1:30,000 dilution. Detection using ProtoGlow ECL or competitor. Blots were exposed for 90 seconds. Lane 1) 100ng; Lane 2) 50ng; Lane 3) 25ng; Lane 4) 10ng; Lane 5) 5ng.

Economical ProtoGlow ECL allows the researcher to use less antibody. The extremely enhanced signal from ProtoGlow ECL allows you to use anywhere from 4 to 40 times less antibody per blot while retaining the same detection sensitivity. This in turn allows you to economize on the consumption of expensive antisera. Sequential exposures of serially diluted HRP conjugated anti-transferrin antibody detected with ProtoGlow ECL or competitor. Exposures of 60 seconds were taken at 0, 30, 60 and 180 minutes. Dilutions: Lane 1) 1:10,000; Lane 2) 1:20,000; Lane 3) 1:40,000; Lane 4) 1:80,000; Lane 5) 1:160,000

AP P L IC ATIO N S Western Blotting.......................... 91 Chemiluminescent Detection.......93

USA: 1-800-526-3867 EUROPE: 441 482 646022

STORAGE: ProtoGlow ECL kit components are best stored refrigerated (4째C). ProtoGlow ECL is stable for up to one (1) year.

Product Name

Cat. No.

Size

ProtoGlow ECL CL-300 200 ml kit 500 ml kit

Electrophoresis Products - Protein Visualization Electrophoresis

Protein Visualization

ProtoBlot Rapid Western Blotting Buffer (10X) TM

l l l

Blot in Half the Time of Conventional Electroblotting Buffers Very High Efficiency Transfer Neutral pH

Simple and effective, the ProtoBlot Rapid Western Blotting Buffer halves the time needed to blot your gels. Blots that normally take an hour to complete can now be done within 30 minutes. Less heating occurs, and the composition is less harsh, leading to better preservation of epitopes. The efficiency of transfer is very high, improving the signal/noise ratio. Product Name

Cat. No.

Size

ProtoBlot Rapid Western Blotting Buffer (10X)

EC-878

1 L

APP L IC A TION S Western Blotting.......................... 91 Colloidal Coomassie Staining....86 Wet Transfer.................................92

ProtoLift Western Stripping Buffer TM

l l l

Strip PVDF Blots in 10 minutes Contains Zero Harsh Detergents Non-Acidic

The ProtoLift Western Stripping Buffer is a more effective system for stripping your PVDF Western Blots. Antibodies can be stripped from blots within 10 minutes. Target proteins are not stripped from the membrane, so blots can be stripped with ProtoLift Western Stripping Buffer multiple times without loss of signal. Note: ProtoLift Western Stripping Buffer is not recommended for nitrocellulose blots.

APP L IC A TION S Western Blotting.......................... 91 Colloidal Coomassie Staining....86

Product Name

Cat. No.

Size

ProtoLift Western Stripping Buffer EC-889

100 ml

Stripping blots..............................93

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis

Protein Visualization

Electrophoresis Products - Protein Visualization

Protein Blotting Buffers

TBS (10X) Tris Buffered Saline

Ultra pure, clear, autoclaved RNase free solution. After dilution to 1X, TBS has the following concentrations: 25 mM Tris; 138 mM NaCl and 2.7mM potassium chloride. Product Name TBS (10X)

TBST (10X) Tris Buffered Saline w/ Tween-20

Size

Same outstanding quality as our TBS buffer with added Tween-20 component. When diluted to 1X, TBST has the following concentrations: 25 mM Tris, 138 mM NaCl, 2.7mM potassium chloride and 0.05% Tween-20. Product Name TBST (10X)

ProtoBlock System

Cat. No.

EC-881 1 Liter

l l

Cat. No.

Size

EC-882 1 Liter

Protein Blocking Solution for Immunoassays Eliminates Endogenous Background

ProtoBlock solution contains a broad spectrum of proteins, protein analogs, detergents, and buffers which are designed to minimize endogenous backgrounds. ProtoBlock solution may be used for Western Blotting, Southern Blotting, immunoassays, and in situ hybridization. Product Name

Cat. No.

Size

ProtoBlock System CL-252 1 System

Tris-Glycine Electroblotting Buffer (10X) AP P L IC A TION Western blotting ......................... 91

Tris-Glycine Electroblotting Buffer (10X) is a concentrated solution for use as the tank buffer in Western electroblotting procedures. Each bottle of Tris-Glycine Electroblotting Buffer (10X) contains 0.25M Tris base and 1.92M glycine. To prepare 1 liter of working strength buffer, add 100 ml of Tris-Glycine Electroblotting Buffer to 200 ml of methanol and 700 ml of distilled/ deionized water. Product Name

Cat. No.

Size

Tris-Glycine Electroblotting Buffer(10X) EC-880 1 Liter 4 Liters (1-3) 4 Liters (4 +)

PBS(10X) Phosphate Buffered Saline AP P L IC A TION Western blotting ......................... 91

USA: 1-800-526-3867 EUROPE: 441 482 646022

Produced as a 10X concentrate, National Diagnosticsâ&#x20AC;&#x2122; PBS is a clear, RNase free solution. After dilution to 1X, PBS has the following electrolyte concentrations: 137 mM NaCl; 2.7 mM KCl; 10mM Phosphate Buffer. Product Name

Cat. No.

Size

PBS(10X) CL-253 450 ml 1 Liter 4 Liters

Electrophoresis Products - Protein Visualization

kDa

190

86 70 47

Proteins Stained with High Definition Blue Dye Red Protein Band Included for Easy Orientation High Contrast, High Intensity Labeling

National Diagnostics’ ProtoMarkers consist of seven (7) purified proteins. Six markers are permanently labeled with high-contrast blue dye. One protein is labeled with high-contrast red dye to facilitate accurate positioning on the gel. ProtoMarkers protein standards range in size from approximately 20 kD to 190 kD, covering the most common protein molecular weights.

29 22

APPL IC ATIO N S 2-dimensional

Gel electrophoresis of

ProtoMarkers are supplied in quantities of 0.5 ml per vial. Each vial contains sufficient material for 100 mini-gels.

proteins........................................69 electrophoresis ............................79

Product Name

Cat. No.

Size

Molecular wt. determination......72 Western blotting ......................... 91

ProtoMarkers

EC-898

0.5 ml

ProtoMetrics

kDa 225

150 100 75 *50 35 25

Electrophoresis

ProtoMarkers

Protein Visualization

Protein Standards

*Gel run using 5 microliters of ProtoMetrics, stained with Coomassie Blue. Note the high intensity 50kD reference band.

Engineered for Exact Mobilities Sharpest, most precise bands for electrophoresis and blotting

National Diagnostics’ ProtoMetrics Protein Markers consist of 9 precisely sized recombinant proteins of 10, 15, 25, 35, 50, 75, 100, 150, and 225 kDa. The ProtoMetrics Protein Markers are supplied in quantities of 0.5 ml per vial. Each vial contains sufficient material for 100 mini-gels.

APPL IC ATIO N S 2-dimensional

Gel electrophoresis of

Product Name

Cat. No.

Size

ProtoMetrics

EC-899

0.5 ml

proteins........................................69 electrophoresis ............................79 Molecular wt. determination......72 Western blotting ......................... 91

Insite Markers

Two Sets of Markers in One

Prestained Markers Provide Orientation During the Run

Engineered Molecular Weight Standards Appear in Fluorescent Detection

kDa 225 150 100 75

kDa 190 86 59

35 35

With fluorescent detection visible markers disappear and engineered standards come forward

APP L IC ATIO N S Gel electrophoresis of

2-dimensional

National Diagnostics’ Insite Markers contain both visible markers for orientation during the run and high precision protein standards (10 - 225 kD) that appear with fluorescent detection. This allows both confident monitoring of the run and precise assignment of protein molecular weights. The Insite Markers are supplied in a 0.5 ml vial. Each vial contains sufficient material to run between 50 and 100 mini-gels.

proteins........................................69 electrophoresis ............................79

Product Name

Cat. No.

Size

Molecular wt. determination......72 Western blotting ......................... 91

Insite Markers

EC-897

0.5 ml

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis

Sample Preparation

Electrophoresis Products - Protein Sample Preparation

Preparing Protein Samples for PAGE ProtoGel Sample Prep Kit

l l l l

Removes interfering contaminants Concentrates dilute samples Prevents gel failures Simple and Inexpensive

Purification Contaminants in the sample such as high salt or urea lead to blurred bands or smiling gels in SDS-PAGE. With the ProtoGel Sample Prep Kit, interfering substances from upstream applications can no longer gain entry to the well. Contaminants are washed away with a simple method. The sample loaded contains only pure protein and loading buffer with no contaminants remaining to impede reproducible, high-resolution results.

Concentration Unpurified and purified samples in SDS-PAGE.

Lanes 1 and 3 are unconcentrated. Lanes 2 and 4 are concentrated.

ND Protein Precipitation Kit

In addition to purification, proteins previously too dilute for SDS-PAGE can now be concentrated prior to electrophoresis with a simple method. The ProtoGel Sample Prep Kit concentrates proteins as dilute as 25ng/100Âľl. The patented ProtoGel Sample Prep Kit casts the finest net of any recovery system, concentrating all proteins in high yield regardless of identity. With the ProtoGel Sample Prep Kit, the purity and concentration of your SDS-PAGE samples are both under your control. Product Name

Cat. No.

Size

ProtoGel Sample Prep Kit

EC-884

1 Kit

Rapid Recovery and Concentration of Proteins l Recovery of All Proteins from Complex Mixtures l Precipitates as Little as 100ng of BSA at 0.25Îźg/ml More effective and much more universal than other methods, the ND Protein Precipitation Kit represents a major breakthrough in the recovery of proteins from complex solutions. (Patent Pending)

APPL IC ATIO N S Sample Preparation.................... 69

USA: 1-800-526-3867 EUROPE: 441 482 646022

The ND Protein Precipitation Kit is universal, mild and easy to use. The ND Protein Precipitation Kit allows the high yield collection of all proteins in solution. Additionally, it casts the finest net of any procedure, precipitating even the most dilute proteins and recovers proteins that would be missed by other methods. Product Name

Cat. No.

Size

ND Protein Precipitation Kit

EC-888

1 Kit - Precipitates 50 ml

Electrophoresis Products - DNA/RNA Visualization

l l

UV Free Visualization of DNA and RNA Improves both Sample Integrity and Yield in PCR Purification from Agarose Gels Positive Stain/High Sensitivity Easy to Use

DNA Visualization

Nuclistain

Electrophoresis

Visualizing DNA and RNA

National Diagnostics offers an improved method of nucleic acid visualization with the development of Nuclistain. Nuclistain is a positive stain concentrate for the rapid detection of double and single stranded DNA and RNA in agarose and polyacrylamide gels. Nuclistain is intended as a replacement for the conventionally used ethidium bromide. Its sensitivity is almost comparable to that of ethidium bromide, with the capability of detecting as little as 10-50 ng of nucleic acid sample. The advantage of Nuclistain is that the results are visible under normal lighting conditions, eliminating the possibility of sample damage by UV radiation. Furthermore, compared to ethidium bromide, Nuclistain visualization dramatically improves the yield of smaller and medium sized DNA (<500 bp) obtained by purification from agarose. Nuclistain binding is reversible and does not modify nucleic acids.

APP L IC A TION S Staining of nucleic acids ........... 81

Nuclistain is extremely easy to use. Simply pour and dilute. One 25 ml bottle of Nuclistain will make 2.5 liters of stain solution. The separated DNA appears as dark blue bands on a light blue background. Product Name

Cat. No.

Size

Nuclistain EC-730 25 ml (1-3) 25 ml (4+)

USA: 1-800-526-3867 EUROPE: 441 482 646022

DNA Visualization

Electrophoresis

Electrophoresis Products - DNA/RNA Visualization

Autofluor 3

H C 14

PPO-DMSO Autofluor

The gel (7%, 1mm) in the illustration at left was dehydrated in DMSO (dimethylsulfoxide) for one hour, then impregnated in PPO-DMSO for one hour and precipitated and dried. The right gel was impregnated with Autofluor for one hour and dried. Both gels were exposed for 24 hours at -76째C on Kodak XR-5 X-OMat film. The single tritiated band contains 5000 dpm. Note the higher degree of resolution and band discrimination with Autofluor vs PPO-DMSO.

APPL IC A TION S Autoradiography ........................85 Staining of nucleic acids ........... 81 Southern blotting ........................84 Staining proteins in gels..............86 Northern blotting ........................83

High Resolution Autoradiographic Image Intensifier l Rapid enhancement of low energy beta-emitters such as 3H, 14C, and 35S l For polyacrylamide gels, paper chromatography, and TLC plates l Water based, odorless, contains no DMSO Autofluor represents the first water soluble scintillation phosphor to be developed and applied directly for use as an autoradiographic image intensifier. Autofluor rapidly penetrates acrylamide gel systems and maximizes energy transfer from labeled compound to phosphor. Autofluor contains no dimethylsulfoxide or aromatic solvents. Therefore, the hazards of use related to these materials are eliminated. The band distortion that is associated with using nonaqueous enhancers is also eliminated. The Autofluor procedure is the shortest and easiest procedure yet developed for enhancement and visualization of beta-emitters. In an independent test1 comparing eight different fluorographic methods for the detection of 35S-labeled proteins in polyacrylamide gels, Autofluor was the most effective. With Autofluor, the dpm/mm2 required to half-saturate the x-ray film was 1/8 that required by autoradiography alone.

Product Name

Cat. No.

Size

Autofluor LS-315 1 Liter (1-3) 1 Liter (4 +)

Bromophenol Blue

Cat. No.

Size

Bromophenol Blue HS-603 10 g

Bromocresol Green

Bromocresol Green is used as a tracking dye for DNA electrophoresis in agarose. Product Name

Cat. No.

Size

Bromocresol Green HS-602 5 g

Xylene Cyanole FF

Xylene Cyanole is a tracking dye for DNA Electrophoresis. Product Name

Cat. No.

Size

Xylene Cyanole FF HS-608 25 g

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Products - Gel Accessories Electrophoresis

Gel Accessories

Gel Accessories National Diagnostics has specialized in providing the best and most convenient electrophoresis reagents for over 25 years. Our unique experience in this field ensures the quality of all materials. Our accessory reagents and supplies are selected and prepared specifically for electrophoretic applications, assuring superior results and reproducibility.

GelDry Film

Glass Bond

GelDry Film is a specially designed drying film to be used in air-drying procedures for polyacrylamide gels. GelDry Film will yield gels that are perfectly preserved, with no wrinkles, pox, or blemishes. Compatible with all current makes of air gel drying systems, GelDry Film is supplied as 11 x 12 cm sheets or 22.5 x 22.5 cm sheets. Product Name

Cat. No.

Size

Gel Dry Film - 11 x 12 cm Gel Dry Film - 22.5 x 22.5 cm

EC-612 EC-622

50 sheets 50 sheets

Glass Bond allows polyacrylamide gels to be affixed to one of the glass plates used for casting electrophoresis gels. The glass plate is chemically modified so that the gel becomes covalently bound to the glass, enabling it to be manipulated without danger of the gel ripping or slipping off the plate. Glass Bond is especially convenient for treating large sequencing plates, as subsequent manipulation can be performed without fear of ripping or disrupting the gel. On plates treated with Glass Bond, the gels dry down to a smooth plastic film without the use of a gel dryer, thus autoradiography can easily be performed. A total of 6.25 liters of bonding solution (enough for over 100 applications on 40 X 40 cm glass plates) can be made from one 25 ml bottle of Glass Bond. Product Name Glass Bond

Glass Free

Size

National Diagnostic’s Glass Free coats glass casting plates for easy release of polyacrylamide gels. Glass Free prevents the cast gel from binding to the removable upper glass casting plate. Glass Free—used in conjunction with Glass Bond—guarantees whole, manageable electrophoretic gels time after time. Glass Free is reusable and has a shelf life of two years. Product Name Glass Free

Ion Exchange Resin (mixed bed) - ULTRA PURE

Cat. No.

EC-620 25 ml

Cat. No.

Size

EC-621 450 ml

Electrophoresis grade Ion Exchange Resin is a mixture of anionic and cationic resins specially prepared for the deionization of electrophoretic gel monomer solutions prior to initiation. o

Wet mesh...............................16-50

Maximum Temperature.......... 60 C

Moisture content...................... 55%

Capacity................... 0.55 meq/ml

Product Name

Cat. No.

Size

Ion Exchange Resin EC-408 100 g USA: 1-800-526-3867 EUROPE: 441 482 646022

Reagents

Electrophoresis

Electrophoresis Products - Reagents

Ultra-Pure Reagents For those cases where our wide array of pre-mixed Electrophoresis solutions do not provide the exact matrix or buffer required, National Diagnostics powdered reagents allow you to formulate your gels with confidence in the purity and reliability of key ingredients. National Diagnostics reagents are manufactured to the most stringently controlled specifications.

Acrylamide -

ULTRA PURE

National Diagnostics’ Acrylamide has been specially purified to produce consistent gel matrices for the most demanding requirements. Reproducible polymerization makes consistent electrophoretic behavior easy to attain. Molecular Weight…………………… 71.08 Purity………………………………… > 99.9% pH…………………………………………… 6.5 Aldehyde Content……………… < 0.001% Acrylic Acid………………………< 0.001% Conductivity (50% solution)………… 20 µmho Acrylamide EC-201 100 g 500 g 1 kg

Aids in formation of networks (high localized concentrations of probes) during hybridization, thus expediting the annealing process. Molecular Weight………………… 500,000

Purity……………………………………… > 99%

Dextran Sulfate EC-877 50 g 250 g

ULTRA PURE

Specially purified of trace metals and other impurities.

Exceeds ACS Standards. Low absorbed water results in consistent initiation. Molecular Weight…………………… 228.2

Purity……………………………………… > 98%

Ammonium Persulfate EC-504 25 g 100 g

Dithiothreitol (DTT) EC-601 1 g 5 g

EDTA -

ULTRA PURE Disodium ethylenediamine tetraacetate dihydrate

BIS - ULTRA PURE

(N,N´ - methylene bisacrylamide) Specially purified crosslinking agent for general electrophoretic use. OD280 (1% solution)………………………… < 0.2 Acrylic Acid………………………… < 0.001% BIS

Purity.................................> 99.9% Conductivity (2% solution)......100 µmho

EC-301

25 g

Molecular Weight…………………… 372.26 Fe………………………………………… < 0.01% Color…………< 10 APHA (0.5M solution) Heavy Metals……………………… < 10 ppm

Purity……………………………………… > 99% Insolubles…………………………… < 0.005% pH (5% solution)………………… < 4.0-6.0

EDTA EC-610 100 g 500 g

Reagent Ethanol ULTRA PURE

Molecular Weight…………………… 61.83 Color……………………………… < 10 APHA SO4…………………………………… < 4 ppm Boric Acid

ULTRA PURE

Dithiothreitol (DTT)

Ammonium Persulfate - ULTRA PURE

Boric Acid -

Dextran Sulfate

ULTRA PURE

Molecular biology grade, denatured ethanol (5% Methanol) pH (1% solution)………………… 5.1 + 0.5 Cl…………………………………… < 0.4 ppm Fe……………………………………… < 2 ppm

EC-609

Reagent Ethanol HS-300 1 Liter 4 Liter 20 Liter

500 g

Formamide - ULTRA PURE Ready-to-use. Deionized and packed under nitrogen.

DATD -

ULTRA PURE (N,N´ - diallyltartardiamide)

Crosslinker with a 1,2-diol structure that may be oxidized by periodic acid. DATD

EC-303

USA: 1-800-526-3867 EUROPE: 441 482 646022

25 g

Molecular Weight…………………… 45.04 Conductivity…………………… < 25 µmho Heavy Metals……………………… < 5 ppm

Purity…………………………………… > 99% Color……………………………… < 10 APHA

Formamide EC-608 200 ml 450 ml

Electrophoresis Products - Reagents

TEMED

Assay………………………………> 99.7% Color……………………………< 10 APHA

Glycerol

EC-606

450 ml

Reagents

(redistilled) - ULTRA PURE (N, N, N´, N´ - tetramethylethylene diamine)

ULTRA PURE

Molecular Weight…………………… 92.09 pH……………………………………… neutral A280…………………………………… < 0.05

Glycine -

Electrophoresis

Glycerol -

Fractionally distilled to remove all trace metals and amine impurities. Boiling Range………………… 119-121C Purity…………………………………… > 99%

Molecular Weight…………………… 16.21 Refr. Index…………… 1.1480 +/– 0.003 TEMED

EC-503

25 ml

ULTRA PURE

Purified by novel chelation chromatography. Molecular Weight…………………… 75.07 OD280 (1M solution)………………… < 0.15 Heavy Metals……………………< 20 ppm Glycine EC-405 250 g 1 kg

Tricine -

ULTRA PURE (N-Tris (hydroxymethylmethylglycine))

Tricine

2-Mercaptoethanol - ULTRA PURE

2-Mercaptoethanol

Water……………………………… 0.5% max Purity…………………………………… > 98% Color……………………… < 30 (max) APHA

EC-603

50 ml

ULTRA PURE Single crystalline species for greater reproducibility.

Riboflavin

Purity…………………………………… > 99% EC-501

25 g

SDS -

ULTRA PURE (Sodium dodecylsulfate)

Molecular Weight………………… 288.38 OD280.(3% solution)……………… < 0.1 Lead (Pb)………………………… < 0.0002%

100 g

ULTRA PURE [Tris(hydroxymethyl)aminomethane] Purified of ammonia and amine contaminants. Molecular Weight…………………121.14 OD280 (1M Solution)………………< 0.15 Mg……………………………………< 1 ppm As……………………………………… < 5 ppm

Purity………………………………… > 99.9% Pb……………………………………… < 1 ppm Fe……………………………………… < 1 ppm Cu……………………………………… < 1 ppm

Tris EC-406 250 g 1 kg

Riboflavin -

Molecular Weight………………… 376.37

EC-407

Tris -

Triple distilled and stored under nitrogen. Molecular Weight……………………… 78.1 Molarity…………………………………… 14.2 Meta-hydroxy-diethyldisulfide… 0.5% max

Purity……………………………………> 99%

Molecular Weight…………………179.17 Heavy Metals……………………< 20 ppm

Tween-20 * - ULTRA PURE Tween-20 EC-607 200 ml 1 liter

Urea Purity……………………………… > 99% FAS C12Sulfate……………………………… > 99% Phosphate (PO4)……………… < 0.0001%

SDS EC-604 100 g 1 kg

SDS Solution (20%) Purified to remove colored contaminants that interfere with spectrophotometric analysis. Zero free sulfates eliminate non-specific binding. SDS Solution (20%) EC-874 100 ml 450 ml

ULTRA PURE

Recrystallized to remove ammonia. Molecular Weight…………………… 60.06 OD280.(5M Solution)……………… < 0.05 Fe……………………………………… < 1 ppm

Biuret test…………………………… negative Heavy Metals…………………… < 0.5 ppm CNO…………………………………< 5 ppm

Urea EC-605 250 g 1 kg

Water - DEPC Treated, Sterile Certified Nuclease Free, Deionized and Autoclaved DNAse……………………………negative RNAse……………………………negative

Conductivity………………< 5micromho Metals……………………………< 5 ppm

Water, Sterile DEPC Treated EC-625 1Liter

USA: 1-800-526-3867 EUROPE: 441 482 646022

Applications

Electrophoresis

Electrophoresis Applications - Fundamental Principles of Electrophoresis

Fundamental Principles of Electrophoresis

1.1 BIOMOLECULES

1.4 BUFFERS

Nucleic Acids / Proteins

Homogeneous Buffer Systems / Multiphasic Buffer Systems / Isotachophoresis / Buffer Additives

1.2 THE MECHANICAL AND ELECTRICAL DYNAMICS OF GEL ELECTROPHORESIS

1.5 THE APPARATUS

Sample Mobility / Electrophoresis System Dynamics

Standard Designs / Running Parameters

1.3 THE MATRIX

Polyacrylamide / Agarose

The Fundamentals of Electrophoresis...Life in the Fast Lane

echniques for separating mixtures of biological substances are of central importance to life science research. Many techniques have been developed taking advantage of differences in chemistry, biology, charge, or size to separate the molecular forms found in biological samples. Of these methods, gel electrophoresis has few rivals for the small-scale separation of proteins or nucleic acids. Because of its high resolution and sensitivity, electrophoresis is universally practiced throughout the fields of molecular biology, biochemistry and biology.

At its most fundamental level, gel electrophoresis involves applying an electric field to a mixture of biological molecules which causes them to migrate through a matrix, or gel. For most electrophoresis techniques, separation then occurs on the basis of molecular size. The larger molecules have more difficulty moving through the gel. Mixture components separate into discrete â&#x20AC;&#x2DC;bandsâ&#x20AC;&#x2122;, the smaller forms having more mobility through the gel. Patterns of such bands are then visualized for a variety of analytical purposes. A number of techniques also exist to extract and purify the nucleic acid or protein from an individual band.

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Applications - Fundamental Principles of Electrophoresis

1.1 The Biomolecules

Applications

Electrophoresis

Of the four kinds of biological macromolecules: nucleic acids, proteins, carbohydrates, and lipids, electrophoresis is generally employed to analyze nucleic acids or proteins. In order to understand their behavior in electrophoresis systems, it is necessary to have a firm grasp of the structure of these molecules.

hydrogen bonding symmetries. The nitrogen base adenine “base pairs” with thymine (or uracil in RNA). Guanine “base pairs” with cytosine. Because of base pairing, DNA or RNA can exist as single stranded or double stranded variants. The double stranded form consists of two complementary strands joined by base pairing.

1.1.1 Nucleic Acids Like many biological molecules nucleic acids are polymers, long molecules formed of repeating units. With nucleic acids, the repeating unit is the nucleotide. A nucleotide consists of a five carbon sugar, a nitrogen containing base and a phosphate group. The two primary kinds of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess slightly different sugars in their respective nucleotides and a different set of four bases which may be contained by their nucleotides.

DNA nucleotide

Figure 1.1.1f The base pairing of two complementary strands allows nucleic acid molecules to assume a double stranded form.

Base pairing can also occur in single stranded DNA or RNA. A section containing one sequence of nucleotides will often loop back and base pair with a complementary section on the same chain. This will affect the 3 dimensional structure of the molecule, with implications for electrophoretic separations. In general, long strands of DNA or RNA will be found in a base-paired conformation, either double stranded or single stranded with internal pairing. Unpaired, or “denatured” nucleic acids are only found in solution under special conditions which destabilize the base pairs.

RNA nucleotide

Figure 1.1.1a The nucleotides of DNA and RNA.

adenine

thymine uracil (DNA ONLY) (RNA ONLY)

guanine

Figure 1.1.1g Base pairing is not limited to double stranded variants, but can also occur within the same molecule. The resulting conformations can lead to electrophoresis results that are difficult to interpret.

cytosine

Figure 1.1.1b The set of bases which may be presented in the nucleotides of DNA and RNA.

Figure 1.1.1c The structure of a section of an RNA molecule.

Of great importance to electrophoresis is the ionization of the phosphate groups, giving nucleic acids a large net negative charge. Because each nucleotide is ionized, the charge to mass ratio of two different nucleic acid molecules will very closely agree.

Figure 1.1.1d Because each nucleotide carries a negative phosphate group, the charge to mass ratios of different nucleic acid molecules are nearly identical.

Figure 1.1.1e Because DNA molecules are negatively charged, electric force causes DNA to migrate toward the positive pole.

DNA and RNA each contain four possible nucleotides corresponding to the set of four possible bases (adenine, guanine, thymine and cytosine for DNA; adenine, guanine, uracil, and cytosine for RNA). Each base exhibits a particular affinity for one of the other three bases, based on

Electrophoresis of double stranded DNA or RNA is referred to as native gel electrophoresis. Electrophoresis of single stranded DNA or RNA occurs under denaturing conditions. Formamide and urea are the two most common agents which accomplish chemical denaturation. These substances act to disrupt the hydrogen bonding between the nitrogen bases, thereby removing the effects of base pairing. Usually some combination of formamide, urea, and heat is employed over the process of denaturing electrophoresis from sample preparation to running the gel. The purposes of denaturing conditions are to ensure single stranded molecules and to prevent conformational changes due to base pairing between different sections of the same DNA or RNA molecule. Denaturing electrophoresis conditions allow for a consistent relationship between molecular size and mobility through the gel.

Figure 1.1.1h Formamide and urea accomplish the denaturation of DNA or RNA by forming new hydrogen bonds with the bases of the nucleic acid molecules, disrupting the hydrogen bonds that lead to base pairing.

Denaturing Agents Formamide EC-608 Deionized and packed under nitrogen. Readyto-use. (pg. 32)

Urea EC-605 Lowest possible ammonia content enhancing long term stability. (pg. 33)

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Applications - Fundamental Principles of Electrophoresis

Like nucleic acids, proteins are polymers. While with nucleic acids the repeating unit is the nucleotide, with proteins, the analogous repeating unit is the amino acid. Amino acids consist of a central carbon which carries an amino group, a carboxyl group, a hydrogen, and a side chain group. Amino acids are distinguished by the properties of their side chains.

Applications

Electrophoresis

1.1.2 Proteins

A protein’s state of ionization depends on the nature of its amino acids and the chemical environment. In neutral, aqueous solution (pH = 7), a protein with a preponderance of basic amino acids, lysine, arginine, or histidine, will have an overall positive charge. Conversely, a protein with many acidic amino acids, glutamic acid or aspartic acid, will have an overall negative charge in neutral solution.

Figure 1.1.2a Amino acids are the basic structural units of proteins. An amino acid consists of an amine group, carboxyl group, hydrogen atom, and a side-chain group bonded to a central carbon atom.

side chain

Figure 1.1.2d A protein with many basic side chains will have a positive charge at physiological pH.

Nonpolar, Non-ionizable Alanine

Leucine

Valine

Phenylalanine

Isoleucine

Proline

Methionine

Tryptophan

Figure 1.1.2e A protein with many acidic side chains will have a negative charge at physiological pH.

Because the state of ionization depends on the pH of the environment, almost all proteins placed in a basic environment will accrue a negative charge, losing hydrogen ions as a function of acid/base equilibrium. A protein placed in an acidic environment will tend to become positively charged. Nondenaturing protein electrophoresis is generally carried out in a weakly basic environment. In this environment, most proteins will become negatively charged and migrate towards the positive plate. Denaturing protein electrophoresis, in the presence of sodium dodecyl sulfate (SDS), also causes proteins to obtain a negative charge through emulsification by negatively charged dodecyl sulfate ions (see section 1.4.4, surfactants).

Polar, Non-ionizable Glycine

Glutamine

Serine

Threonine

Tyrosine

Asparagine

Basic Arginine Lysine

Figure 1.1.2b side-chains.

Cysteine

Acidic Histidine

Aspartic acid

Figure 1.1.2f Emulsification by sodium dodecyl sulfate gives proteins a net negative charge. Different proteins in the same SDS solution are imparted with approximately the same charge to mass ratio, an advantage of SDS-PAGE electrophoresis.

Glutamic acid

Amino acids are classified according to the properties of their

The pH where a protein is electrically neutral overall is a function of the type and number of the protein’s ionizable groups. At this pH, called the isoelectric point (pI) of the protein, it will not migrate in an electric field. Because the distribution of ionizable groups is different among proteins, they differ in their isoelectric points. This difference is a powerful tool for electrophoretic separation, used in isoelectric focusing (see section 3.3.1).

Single chain proteins generally range from 50 to 1000 amino acids in length. When describing protein structure, biologists distinguish primary, secondary, tertiary, and quaternary levels of structure. A protein’s primary structure is the actual sequence of amino acids. The secondary structure refers to local bends, kinks and spirals along the chain. Tertiary structure refers to the shape of the entire polypeptide chain, and quaternary structure is used to describe proteins which consist of more than one polypeptide chain.

Isoelectric Point

Figure 1.1.2g A protein’s state of ionization is determined by the nature of its side chains and the pH of the solution environment. At the pH that is its isoelectric point, a protein has equal numbers of positive and negative charges and does not migrate in an electric field.

SDS 20% Solution or ULTRA-PURE Powder primary structure

Figure 1.1.2c

secondary structure

tertiary structure

The levels of protein structure. USA: 1-800-526-3867 EUROPE: 441 482 646022

quaternary structure

SDS Solution 20% EC-874 Saves time and provides more reproducibility than making up SDS solution in the lab. (pg.

33)

SDS - ULTRA PURE EC-604 Guaranteed free of sulfates and colored contaminants. (pg. 29)

Electrophoresis Applications - Fundamental Principles of Electrophoresis

1.2 The Mechanical and Electrical Dynamics of Gel Electrophoresis

Within an electrophoresis gel, a protein or nucleic acid molecule experiences electric force proportional to its effective charge, Q, and the electric field strength, E . The biomolecule will move with a constant velocity because the electric force on the molecule is met by an opposite force due to the frictional resistance, f, of the matrix.

Applications

1.2.1 Sample Mobility

1.2.2 Electrophoresis System Dynamics

Electrophoresis

The term ‘electrophoresis’ refers simply to the movement of particles by an electric force. The first electrophoresis experiments were carried out on molecules in a conductive buffer solution, where the only force acting on the sample was the electric field. The vast majority of current laboratory techniques are performed in a matrix perfused by an aqueous solution of buffer salts. The addition of a matrix (or gel) stabilizes the system against solution turbulence, and extends the range of geometries available to the user. The primary purpose of the matrix, however, is to introduce a sieving action which allows separations based on molecular size.

effect on the migration of sample molecules. Higher ionic strength in the solution reduces the effective charge of the sample molecules. Therefore, low ionic strength solutions generally correspond to high rates of migration. Electrophoresis in higher ionic strength solutions generally gives slower rates of migration but produces sharper bands. A problem occurs, however, with high ionic strength buffers, when excessive power generation causes the gel temperature to increase. This temperature increase leads to a decrease in the viscosity of the medium, which then leads to increased current and further heating. Buffer evaporation, sample denaturation, and convective mixing are typical problems which can result in this scenario.

The apparatus in gel electrophoresis constitutes an electrical/thermodynamic system. The apparatus receives energy from the power source and releases energy as heat. The figure below shows a stylized representation of a typical vertical slab gel apparatus. The gel, perfused with buffer solution and held between two glass plates, has been clamped in position, its upper end is immersed in the buffer solution of the upper electrode chamber. The lower end of the gel is immersed in the lower electrode chamber, which contains buffer solution as well.

If electrophoresis occurred in free solution, rather than within a gel, the force of frictional resistance, f, upon biomolecules would follow Stokes’ law: f = 6πrυη where r is the radius of the particle moving with velocity υ through a medium of viscosity η. The electromotive force on a molecule, proportional to its charge, is thus opposed by the frictional force f , proportional to its mass. Mobility in free solution would then be the same for molecules of the same charge to mass ratio. Stokes’ law, however, is not sufficient to describe the frictional force within a gel matrix. In addition to the viscosity of the medium, also determining frictional resistance are the density and effective “pore size” of the matrix. The result of this combination of factors is that among molecules of the same charge to mass ratio, larger molecules move more slowly in gel electrophoresis and electrophoretic separation occurs by size. In techniques such as denaturing DNA or RNA electrophoresis or SDS-PAGE protein electrophoresis, conditions are maintained so that the charge to mass ratio is virtually equal among sample molecules and separation occurs in a predictable manner based on the size of the molecules.

v v v

Figure 1.2.1a The gel electrophoresis of samples having identical charge to mass ratios results in fractionation by size. Although experiencing greater electric force because of their greater charge, the larger molecules reach force equilibrium with the opposing frictional resistance at a slower speed, thus their lower electrophoretic mobility.

Two additional factors determining sample mobility are the ionic strength of the surrounding solution and the temperature of the gel. To maintain physiological pH and control the net charge on biological molecules in electrophoresis, the solution perfusing the gel matrix contains buffer ions. The ionic strength of this buffer will have a strong

Figure 1.2.2a A gel electrophoresis apparatus is essentially a resistor (the gel) connected to a voltage source, creating a simple DC circuit. The voltage drop across the gel provides the motive force which drives the buffer and sample ions through the gel.

After samples are introduced into the sample wells at the top of the matrix, the electric field between the electrode chambers causes them to migrate toward the lower chamber. The electrophoresis apparatus can be viewed as a simple DC circuit composed of one voltage source and three resistors in series, the resistors being the upper chamber, the gel, and the lower chamber. Because the cross-section of each electrode chamber is much greater than the cross-section of the gel slab, and because the chambers are shorter in length as well, the vast majority of the resistance in the circuit derives from the gel slab. For this reason it is sufficient to consider the gel slab as the only resistor in the circuit, where the vast majority of power is expended. An exception to this rule occurs if buffer salts are inadvertently absent from one or both of the electrode chambers.

Ohm’s Law Relates the Electrical Parameters

Ohm’s law describes the relationship between the voltage, V, the current, I, and the resistance, R, in a DC circuit. A greater voltage produces a greater proportional current through a given resistor: V=IR A variation of Ohm’s law describes how small changes in electrical current can produce large changes in the expenditure of power, P: P=IV =I2R USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Applications - Fundamental Principles of Electrophoresis

Applications

Electrophoresis

Although the electrical current through the gel consists of both migrating buffer ions and sample molecules, the vast majority of the current is represented by the buffer ions. As voltage is applied, the cations in solution migrate toward the negative electrode in the upper chamber, and the anions (and negatively charged sample molecules) migrate toward the positive electrode in the lower chamber. Several factors must be in balance for gel electrophoresis to proceed toward good results. Of major concern is the management of the heat generated by current flow. While small buffer ions, for example, might be more effective in preventing interactions among sample molecules, very mobile ions create a much more conductive buffer. Excessive current flow will be accompanied by excessive heat generation, which can cause convection currents, solution evaporation, or in the case of agarose electrophoresis the melting of the matrix itself. The management of heat is a major reason for the choice of bulky, organic ions, such as Tris base or glycine. (See section 14 for a more detailed treatment of electrophoresis buffers.) Because temperature regulation is especially critical in both polyacrylamide and agarose electrophoresis of DNA and RNA, this type of electrophoresis is most often carried out under conditions of constant power (or constant current with agarose electrophoresis, for historical reasons). In denaturing PAGE electrophoresis, a relatively high temperature must be established and maintained to prevent the renaturation of sample molecules, but the temperature cannot be allowed to get too high. Constant power conditions (or constant current) provide the most precise regulation of heat generation. A small variation in buffer concentration can be managed under conditions of constant power but would lead to a total failure under conditions of constant voltage. Constant voltage conditions, however, are generally employed in the SDS-PAGE electrophoresis of proteins. Here, the generally smaller apparatus has less difficulty exchanging heat with the environment, and sample denaturation is not nearly as temperature sensitive. In this scenario, the advantages of directly controlling the field strength via constant voltage conditions outweigh those of directly controlling the power generation.

1.3 The Matrix In gel electrophoresis the matrix forces sample components to separate by size, as they move through its porous structure. The matrix provides greater resistance to the movement of larger molecules. It also performs additional functions including the reduction of convection currents and the inhibition of sample diffusion, encouraging the separated components to remain as sharp, distinct bands. Furthermore, the matrix serves as a solid medium upon which samples can be fixed and detected in post-electrophoretic analysis. A suitable matrix should be chemically inert during electrophoresis. It should be easy to prepare and to modify. Two materials, polyacrylamide and agarose, have proven themselves to satisfy these requirements very well, performing all the above functions, each having advantages within a specific set of applications.

1.3.1 The Polyacrylamide Matrix Polyacrylamide gels are formed by the polymerization of acrylamide in aqueous solution in the presence of small amounts of a bifunctional crosslinker. The crosslinker is usually methylenebisacrylamide (bis, or MBA). The copolymerization of acrylamide with methylenebisacrylamide produces a mesh-like network in three dimensions, consisting of acrylamide chains with interconnections formed from the methylenebisacrylamide. A variety of crosslinkers are available in addition to bis. These include piperazine diacrylate (PDA), N,N’-bisacrylylcystamine 38

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(BAC), and N,N’-diallyltartardiamide (DATD). PDA is used to reduce silver stain backgrounds in SDS-PAGE gels. BAC and DATD are both disruptable cross-linkers which enable gels to be solubilized. Ammonium Persulfate

TEMED Acrylamide

N,N´- methylenebisacrylamide

The Polyacrylamide Matrix Figure 1.3.1a The polymerization of a polyacrylamide matrix with methylenebisacrylamide cross-linking.

For discussions of the composition of polyacrylamide gels, a standard nomenclature has been widely adopted. In this nomenclature, T represents the total percentage concentration (w/v) of monomer (acrylamide plus crosslinker) in the gel. The term C refers to the percentage of the total monomer represented by the crosslinker. For example, an 8%, 19:1 (acrylamide:bisacrylamide) gel would have a T value of 8% and a C value of 5%. Upon the introduction of catalyst, the polymerization of acrylamide and methylene bisacrylamide proceeds via a free-radical mechanism. The most common system of catalytic initiation involves the production of free radicals by ammonium persulfate in the presence of the tertiary aliphatic amine N,N,N’,N’-tetramethylethylenediamine (TEMED). Another catalytic system involves the generation of free radicals via a photochemical process using a very small amount of riboflavin in the presence of TEMED. In both catalytic systems, the presence of excess oxygen will inhibit the polymerization elongation process and can lead to shorter average chain length. For this reason, if the casting solution has been excessively agitated, deaeration under vacuum with a magnetic stirrer is suggested prior to addition of initiators. For certain applications, polyacrylamide has definite advantages compared to agarose. In an agarose gel, the pore size is large, so molecular sieving, i.e. separation by size, will not occur for smaller DNA fragments and most proteins. Additionally, by altering the total concentration of monomer in the gel and the ratio of acrylamide to bis, the pore size with a polyacrylamide gel can be altered in a reproducible manner. The small and reproducible pore size in polyacrylamide gels results in superior resolution: a 0.1% difference in size (1 base difference in a 1kb molecule) can be detected. Also, because acrylamide and bis are synthetic chemicals, there are virtually no batch to batch differences (It should be mentioned that batch to batch differences with agarose are overcome with the highest quality agaroses, such as National Diagnostics’ AquaPor agaroses). Control of the pore size of a polyacrylamide gel is accomplished by changing the T and C values. With increasing T, the pore size decreases in a nearly linear relationship. Higher percentage gels (higher T), with smaller pores, are used to separate smaller molecules. The relationship

Electrophoresis Applications - Fundamental Principles of Electrophoresis of C to pore size is more complex. Generally, the minimum pore size occurs when C is about 5% (a 19:1 gel). Decreasing C results in a more open pore structure because there are fewer crosslinker molecules. Increasing C beyond 5% also increases the pore size. This appears to be because of nonhomogeneous bundling of strands in the gel.

Applications

A natural colloid extracted from seaweed, agarose is a linear polysaccharide made up of the repeating unit agarobiose, which consists of alternating units of 1,3-linked β-D-galactopyranose and 1,4-linked 3,6-anhydro-α-L-galactopyranose. Gels prepared from agarose have a substantially larger pore size than polyacrylamide gels. Agarose gels can be prepared faster than polyacrylamide but have lower resolution, especially for smaller molecules. For this reason, agarose gels have limited application for the electrophoresis of small nucleic acid fragments and most proteins. However, for the electrophoretic separation of larger nucleic acid molecules (>400 bp), agarose presents an excellent choice. It is easier to handle and safer to prepare than polyacrylamide.

Electrophoresis

Researchers have settled on C values of 5.0% (19:1 acrylamide/bis) for most forms of denaturing DNA and RNA electrophoresis and 3.3% (29:1) for most native DNA and RNA gels. For SDS-PAGE electrophoresis of proteins, the standard C value that has been adopted is 2.6% (37.5:1). The table below gives recommended acrylamide/bis ratios and gel percentages for different molecular size ranges.

1.3.2 The Agarose Matrix

Polyacrylamide Gel Applications

Recommended applications for each formulation are shown in bold

Acrylamide:MBA Ratio

Gel %

Native DNA/RNA (bp)

Denatured DNA/RNA (bp)

Protein (kd)

19:1

100-1500

70-500

100-200

“

60-600

40-400

40-150

“

40-500

20-200

20-100

“

30-300

15-150

15-70

“

20-150

10-100

8-60

29:1

200-2000

70-800

>150

“

80-800

50-500

50-200

“

60-400

30-300

30-125

“

50-300

20-200

20-100

“

40-200

15-125

10-70

“

<40

<30

37.5:1

60-200

“

50-150

“

25-100

“

15-80

Table 1.3.1a

Agarobiose Figure 1.3.2a Agarobiose is the basic unit of the agarose polymer.

A potentially significant problem in agarose electrophoresis is electroendosmosis (EEO). Electroendosmosis in electrophoresis refers to the flow of water under the influence of an electric field due to immobilized charge groups on the matrix, primarily sulfate and carboxyl groups. While the sulfate and carboxyl groups cannot migrate, their counter ions do migrate, moving toward the cathode, causing a net flow of water through osmosis in that direction. In the past, this problem often led to badly smeared bands. Fortunately, purification methods have been developed which nearly eliminate this problem in modern agarose electrophoresis. The use of a high quality electrophoresis grade agarose, such as a member of National Diagnostics’ AquaPor agarose family of products, will prevent electroendosmosis effects. Through the careful modulation of polymer length and helical parameters, the varieties of National Diagnostics’ agaroses are manufactured to display the characteristics suited for different electrophoresis applications.

Matrix formulations for various applications.

Ready-to-Use Acrylamide Products and Catalytic Initiators ProtoGel EC-890 30% solution of Acrylamide and BisAcrylamide, 37.5:1 ratio. Filtered, Deionized, and Stabilized.

UreaGel-6 EC-836 Convenient system for casting 19:1 6% denaturing gels with Acrylamide, Bis, TEMED, TBE and urea. (pg. 12)

AccuGel 19:1 EC-850 40% solution of Acrylamide and BisAcrylamide, 19:1 ratio. Filtered, Deionized, and Stabilized. (pg. 15)

UreaGel Sequencing Sys. EC-833 Three bottle system to conveniently formulate 19:1 acrylamide/bisacrylamide sequencing gels, up to 20%. (pg. 13)

AccuGel 29:1 EC-852 40% solution of Acrylamide and BisAcrylamide, 29:1 ratio. Filtered, Deionized, and Stabilized.

TEMED EC-503 Our TEMED is distilled twice to remove all inhibitory and fluorescent compounds. (pg. 33)

(pg. 8)

(pg. 10)

AcrylaGel EC-810 30% stock solution of Acrylamide. Eliminates the need for handling toxic Acrylamide powder.

Ammonium Persulfate EC-504 >98% pure molecular biology grade. Low absorbed water for consistent polymerization.

Figure 2.4.2a Restriction mapping involves treatment of a DNA fragment with National Diagnostics AquaPor Agarose restriction enzymes both singly and in combination. The electrophoresis of cleavage products yields a map of the DNA in terms of restriction sites.

AquaPor LE EC-202 High quality, general purpose agarose ideal for most routine applications. Low EEO. DNase & RNase free. Unique low boil-over formulation. (pg. 16)

AquaPor 3:1 EC-206 Fine resolution of small DNA fragments. Yields strong gels with low UV background. Low viscosity makes pouring high% gels much easier. (pg. 17)

AquaPor LM EC-204 Low melting agarose. Certified for in-gel ligation and PCR. DNase & RNase free. Highest gel strength available in a low-melting agarose. (pg. 16)

AquaPor HR EC-205 AquaPor HR combines high resolution with low melting. Resolves DNA down to 2% size difference (or 4 bp below 200 bp). DNase & RNase free. (pg. 17)

AquaPor ES EC-203 AquaPor ES is a premium, ultra high strength, ultra low EEO agarose. Ideal for Pulsed Field Gel Electrophoresis (PFGE). (pg. 17)

(pg. 32)

(pg. 11)

Bis-AcrylaGel EC-820 2% stock solution of Bisacrylamide, molecular biology grade. Filtered, Deionized, and Stabilized. (pg. 11)

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Applications

Electrophoresis

Electrophoresis Applications - Fundamental Principles of Electrophoresis

1.4 Buffers

of sample molecules.

In its simplest form, a buffered solution contains a mixture of a weak acid and its conjugate base.

In addition to controlling the pH of the gel, which prevents damage to the sample molecules and controls the ionization state of the molecules, the buffer system also carries the majority of the current through the gel. For a homogeneous system like denaturing PAGE electrophoresis of DNA, in which the type and concentration of buffers in the tank and gel are the same, the buffer prevents wide swings in pH and controls the conductivity of the gel. For the native electrophoresis of proteins, the buffer pH has the added function of controlling the state of ionization of the samples. In this second case, even slight changes in pH can result in large effects on the relative mobility of sample components. In a multiphasic system, such as SDS-PAGE electrophoresis of proteins, where buffers in the tank and gel are different, the considerations of buffer design can take on an even greater level of complexity.

H+ + A-

The position of acid/base equilibrium is represented by the acid dissociation constant, Ka. This number is large if the acid is stronger and equilibrium tends toward dissociation. It is small for an equilibrium that tends toward proton capture. Buffers used in life science tend to range from 10-4 to 10-10 in their Ka values.

Ka =

[H+][A-] [HA]

Ka is usually expressed as its negative logarithm, pKa:

pKa = - log Ka A buffer with a Ka of 10-2 has a pKa of 2, favoring dissociation. A buffer with a Ka of 10-12 has a pKa of 12, favoring proton capture. After a few math operations the expression for the dissociation quotient assumes a very useful form, the Henderson-Hasselbalch equation:

[basic form] pH = pKa + log [acidic form] The formula states that the pH of a buffered solution will differ from the bufferâ&#x20AC;&#x2122;s pKa by an amount which is determined by the ratio of the base to the acid forms in solution. If these concentrations are equal then the pH = pKa. If the concentration of the base form is greater than the acid then the pH > pKa. If the concentration of acid form is greater than the base then pH < pKa. A buffer maintains nearly constant pH by absorbing protons released by other sources in solution or releasing protons if another species is depleting them. For example, a small amount of strong acid introduced to pure water causes the pH to plummet five or six points. However, if that same amount of strong acid is introduced to a concentrated buffer solution, it merely causes the change of some of the bufferâ&#x20AC;&#x2122;s weak base form to the weak acid form. By the Henderson Hasselbalch equation, to drop the pH of the solution a full point below the pKa, enough base would have to be consumed for the concentration of the acid form to become ten times greater than the base. If the buffer is sufficiently concentrated, the small amount of strong acid in this example would barely alter the pH. In electrophoresis, the buffer is able to maintain a relatively constant pH as long as either the acid or the base do not become exhausted. The Henderson-Hasselbalch equation gives information in addition to how the pH is a function of the relative predominance of acid base forms of a buffer. For certain instances, it is useful to think of this equation the other way around. In predicting the state of ionization of minor solution components, for example, it can be useful to think of the relative predominance of various forms as a function of an externally determined pH. An instructive example might be a solution whose pH is maintained by a concentrated buffer. The degree of ionization of a minor component in this solution, such as a protein, would depend on the pH of its environment, determined by the buffer. Suppose an ionizable group upon a protein has a pKa of 11, an amine group, for example, which might be either the nonionized amine form (base) or the ammonium ion form (acid). If the solution is buffered to a stable pH of 8, the Henderson-Hasselbalch equation tells you that the ammonium ion form will predominate at a ratio of 1000 to 1. This concept is essential to understanding how buffers control the state of ionization 40

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In all cases, the ionic strength of the buffer in the gel must be sufficient to keep the sample in solution and to provide sufficient buffering capacity. Higher concentrations of gel buffer will tend to slow the diffusion of samples, and result in sharper bands. The benefits of higher buffer concentrations must, however, be balanced against the fact that the more concentrated the buffer in the gel, the higher the electrical conductivity. With higher concentration, at a given voltage, the current will be greater and more heat will be generated. Because of the problems caused by excessive heating, high buffer concentrations must be accompanied by a low voltage gradient. Several factors to consider when choosing a buffer include: 1) pKa value - A buffer should be chosen with a pKa that is very close to the desired pH, preferably within a half point. The buffer will have the greatest capacity both to absorb or release protons with the acid and the base form well represented in solution. It should be noted that pKa is not constant for all conditions but is a function of the total ionic strength and the temperature, so the stoichiometry should be modeled after actual running conditions. Amines are particularly susceptible to changes in pKa with temperature, because, with amines, there is no net increase in ions in solution with the dissociation of ammonium species, so there is little inherent entropy change due to changes in the ordering of water molecules in the solution. This leads to an increase in the significance of the entropy change due to heat flow (the ratio of enthalpy change to temperature) in determining the position of equilibrium. Usually, in native protein electrophoresis, basic proteins are best separated at acid pH. However, the vast majority of proteins have isoelectric points below 7.5 and are best separated in slightly alkaline conditions, pH 8-9. This pH range has also proven efficacious for most forms of DNA electrophoresis. Thus, buffers with a pKa in the range of 7-9 are best suited for most electrophoretic applications. 2) Formal charges of buffer species - Generally, buffers which form ions of high charge magnitude (+2, +3, -3, etc.) are more difficult candidates with which to work. This type of buffer yields high ionic strength without providing high buffering capacity. At relatively low concentrations, the gel conducts too much current. Furthermore, with ions moving quickly through the gel, the buffer may become depleted. One of the reasons tris-borate is a popular buffer for electrophoresis is that both tris base and borate are uncharged part of the time at the desired pH, which reduces their electrophoretic mobility. Reduced mobility of the buffer ions allows for high concentrations of the buffer solution to be employed with the consequent benefits to buffering capacity and sample stability without producing unacceptably high conductivity. It should be further mentioned that the relative electrophoretic mobility of buffer components is a major concern in the design of multiphasic systems, which are discussed in detail in a following section. 3) Molecular size - In addition to its low charge, Tris base moves slowly in electrophoresis because of its relatively large molecular size. Having a low charge to mass ratio, Tris moves much more slowly than

Electrophoresis Applications - Fundamental Principles of Electrophoresis small ions such as chloride or phosphate. Buffer ions are not sieved by the matrix, so their migration rates are determined solely by their charge to mass ratio.

Commonly Used Electrophoresis Buffers Buffer

Molecular Weight

pKa

∆pKa/°C

Acetic Acid

60.05

4.8

-0.0002

Boric Acid

61.83

9.23

-0.002

Citric Acid

192.1

6.4 (pK3)

Glycine

75.07

9.8

-0.03

MOPS

209.26

7.2

-0.006

Phosphoric Acid

7.2 (pK2)

-0.003

Taurine

125.1

9.1

Tricine

179.18

8.15

-0.021

Tris

121.1

8.06

-0.028

Applications

Figure 1.4.2a A t t h e start of multiphasic SDSPAGE protein electrophoresis the anions are chloride (green) in the stacking and resolving gels and glycine (orange) in the tanks.

Electrophoresis

These three factors are by no means exhaustive. Other factors to consider when choosing a buffer would include toxicity, solubility, UV absorption and the possibility of interaction with other species present in the solution. Among the most commonly used electrophoresis buffers are those shown in the table below:

As electrophoresis begins both Cl- and glycinate ions begin to migrate through the stacking gel. Because the pH is several points lower in the stacking gel than the pKa2 of glycine, the vast majority of glycine molecules are zwitterionic at any moment and their mobility is very low. Because the mobility of the chloride ions is greater than the glycine, the chloride ions (leading ions) begin to migrate away from the glycine (trailing ions). The chloride ions don’t move far before they leave behind an area of unbalanced positive counter ions. A steep voltage gradient develops, the Kohlrausch discontinuity, which pulls the glycine along so that the chloride and glycine ions become successive fronts moving at the same speed. The ion fronts sweep through the sample molecules. Being intermediate in their mobility between chloride and glycine, the sample molecules are carried along becoming ‘stacked’ into very thin, distinct layers in order of electrophoretic mobility.

Table 1.4a Characteristics of buffer salts used in electrophoresis.

1.4.1 Homogeneous Buffer Systems In a homogeneous buffer system, the identity and concentration of buffer components are the same in the gel and the tanks. Most forms of DNA and RNA electrophoresis generally use homogeneous buffer systems. Electrophoresis of proteins is most often performed under multiphasic conditions, where tank and gel buffers differ. In a homogeneous system the buffer performs the functions of protecting the samples and carrying the current. Problems most often arise with homogeneous systems if the ionic strength is for some reason different in the gel versus either of the tanks. Most often this occurs due to the inadvertent omission of a component, a mistake in stoichiometric calculations, or precipitation in a stock solution (10X TBE is especially prone to this problem). One consequence of a concentration gradient between the tank and the gel, is a “salt wave” which migrates through the gel during electrophoresis. Accompanying this concentration gradient will be changes in the electrical parameters leading to localized distortions in the band pattern.

1.4.2 Multiphasic Buffer Systems Employing gel and buffer discontinuities to produce sharp separation among sample components, multiphasic electrophoresis design can improve the resolution of electrophoresis (especially protein electrophoresis). The system employs a separating gel in which the sample is fractionated, above which has been added a low percentage stacking gel. In the stacking gel, sample components are stacked into very thin, sharp zones prior to the final separation. To illustrate the principles of multiphasic systems, we shall examine the most prominent application of this type, the SDS-PAGE electrophoresis of proteins. Multiphasic SDSPAGE protein electrophoresis is often referred to as the Laemmli system after the researcher who perfected the design. At the beginning of the experiment, the buffer compositions are different in the stacking gel, the separation gel, and the tank. The stacking gel contains a Tris-HCl buffer at pH 6.8. The separation gel contains a higher concentration Tris-HCl buffer at pH 8.8. The tanks contain Tris-glycine at pH 8.8.

Figure 1.4.2b With the establishment of the Kohlrausch boundary in the stacking gel, the proteins are stacked into a thin layer between the leading chloride ions and the trailing glycine molecules.

When the interface between the stacking and separation gels is reached by the moving boundary region, the pH changes abruptly (as well as the pore size). At the higher pH a much higher percentage of glycine will be in the ionized state, and its mobility will be increased commensurately. The mobility of sample molecules is decreased by the sieving of the new, higher percentage matrix. The glycine accelerates past the stacked layers of sample molecules and the process of unstacking in the separating gel begins. From this point on, the electrophoretic separation occurs under conditions of constant pH and voltage that are indistinguishable from homogeneous PAGE.

Figure 1.4.2c Electrophoresis in the resolving gel is indistinguishable from ordinary homogeneous buffer systems.

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Electrophoresis Applications - Fundamental Principles of Electrophoresis

Applications

Electrophoresis

1.4.3 Isotachophoresis Isotachophoresis is a method of electrophoresis which employs the basic principles of the stacking gel phase of multiphasic systems discussed in the preceding section. Employing nonsieving media, often low percentage polyacrylamide, isotachophoresis in its simplest form can be thought of as a stacking gel alone, without the separation gel. The principles are the same. With applied electric field, like charged ions begin to separate by electrophoretic mobility. As the faster component begins to move away, a region of unbalanced counter-ions forms behind it. Associated with this region of unbalanced counter ions is a zone of higher voltage, a Kohlrausch discontinuity, which pulls the next fastest component along at the same speed. Although, technically, isotachophoresis separates samples by electrophoretic mobility, the layers of sample molecules move at the same speed. While generally not achieving the resolution of other forms of electrophoresis, isotachophoresis has been successfully employed for difficult samples, such as very small peptides, not amenable to traditional techniques. Isotachophoresis has also shown great promise for the analysis of complex mixtures of molecules of different classes.

1.4.4 Buffer Additives In most forms of electrophoresis the solution perfusing the gel matrix typically contains one or more substances in addition to the buffer salts. Serving the purpose of modifying the properties of sample molecules, these additives can be categorized as hydrogen bonding agents, surfactants, or reducing agents.

Hydrogen bonding agents

Urea or formamide can be introduced into electrophoresis samples prior to loading or into the gel buffer itself in order to cleave hydrogen bonds. These substances disrupt hydrogen bonds by occupying the bonding sites themselves. Hydrogen bonds are dipole-dipole attractions that occur between polar, hydrogen containing functional groups such as amine or hydroxyl groups. Hydrogen bonding has a major influence on the conformation and solubility of biological molecules. It is frequently necessary to include one or both of these substances to standardize sample conformation or to solubilize samples. In denaturing DNA and RNA electrophoresis, formamide plus heat in the sample preparation stage followed by urea in the gel buffer are employed to disrupt the hydrogen bonding relationships central to base pairing. By substituting their own hydrogen bonding relationships with sample molecule functional groups, formamide and urea cause the separation of the complementary strands in double stranded DNA and RNA and, furthermore, disrupt the kinks and loops in single stranded species brought about by self-annealing. The resulting molecules are long and straight, and the influence of small differences in conformation on electrophoretic mobility is minimized. Because of the importance of this technique in electrophoresis, the terms ‘urea gel’ and ‘denaturing gel’ are often used interchangeably in the laboratory. Urea can be employed in protein gels if the sample molecules are insoluble or aggregated, although detergents can also be used. A downside to the use of urea with proteins can be the formation of cyanate ions which will react with some proteins, although Tris buffers will effectively protect the protein samples. If Tris buffers cannot be used, pre-running the gel for 30-40 minutes before adding samples or treating the urea with ion exchange resin before mixing the gel solution can also effectively solve this problem.

Surfactants

A crucial initial step in the electrophoretic separation of proteins is the solubilization of the sample molecules. This is especially true if there are extensive nonpolar interactions. Although urea in high concentration

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was often employed in the past for this purpose, researchers now often have recourse to the use of nonionic, anionic, or cationic detergents.

Figure 1.4.4a SDS is the detergent most commonly employed in protein electrophoresis.

Nonionic detergents, such as Tween-20 or Triton X-100, are generally less strongly denaturing than anionic or cationic detergents. Researchers use nonionic surfactants to preserve enzyme activity or some delicate immunological properties that anionic or cationic detergents would destroy. Generally, Tween-20 or Triton X-100 are added sparingly to the gel buffer (0.1%). These substances can also be employed in a 1% solution for the pretreatment of samples. A major drawback with nonionic detergents is that, unlike charged surfactants, these detergents produce no consistent charge to mass ratio among sample molecules for electrophoresis. For this reason, the molecular weight of proteins cannot be directly determined by one electrophoretic run. In general, electrophoresis results are more difficult to interpret than results from electrophoresis that has been carried out in the presence of charged surfactants such as SDS (sodium dodecyl sulfate). By far the most commonly employed detergent additive in protein electrophoresis is the anionic surfactant SDS (sodium dodecyl sulfate). Proteins under treatment with SDS become completely blanketed by negatively charged dodecyl sulfate anions, unwinding to assume an extended conformation. The number of bound detergent molecules is quite large, approaching half the number of amino acid residues. As a result, the intrinsic charge of treated proteins becomes overwhelmed by the charge of the surfactant molecules, and even proteins of widely divergent structure have a virtually uniform charge to mass ratio. Electrophoresis of such samples results in strict separation by molecular weight. Protein electrophoresis using SDS is treated in depth in section 3.1. Although rarely necessary, cationic surfactants such as CTAB, cetyltrimethylammonium bromide, can be used for the electrophoresis of samples posing difficulties for SDS-PAGE. Such cases include the electrophoresis of either extremely acidic or extremely basic samples. Being very negatively charged, extremely acidic samples can exhibit poor binding with SDS. The problem with extremely basic samples is that addition of SDS can lead to precipitation. The use of CTAB as an alternative carries the same benefit of SDS in that a uniform charge to mass ratio among sample molecules is produced, although the apparatus will need to be adjusted to allow samples to migrate toward the negative pole rather than the positive pole.

Reducing Agents

Disulfide bonds between or within sample protein molecules can lead to the formation of aggregates as well as play a role in the binding of the subunits of many proteins. It is usually desirable to cleave disulfide linkages prior to the protein electrophoresis. For this reason, disulfide bond reducing agents, such as 2-mercaptoethanol or dithiothreitol, are typically present in sample buffers. These substances can also be added to the cathode tank. However, 2-mercaptoethanol or dithiothreitol are typically not added to the gel casting solution because their presence inhibits gelation.

2-Mercaptoethanol Dithiothreitol Figure 1.4.4b 2-Mercaptoethanol or Dithoiothreitol are often employed in protein sample buffers to cleave disulfide linkages.

Electrophoresis Applications - Fundamental Principles of Electrophoresis

1.5 The Electrophoresis Apparatus

In its simplest form, a horizontal gel apparatus consists of a box which is divided into two compartments by a platform in the middle (Figure 1.5.1a). The gel is placed on this platform, and buffer is added until the gel is fully submerged. Electrodes in each compartment supply the electric field. The resulting current flows through both the gel and the buffer over the gel, so the thickness of these must be controlled for fully reproducible results. Cooling is provided by the buffer which surrounds the gel, and this buffer is often recirculated to prevent the development of a pH gradient and also to aid in temperature control. Access to the gel is through the overlaying buffer. Samples are loaded through this buffer layer, and the apparatus has a clear lid to allow the run to be monitored. One major limitation of the horizontal apparatus is that, since the two compartments are connected by a layer of buffer, it is not possible to use discontinuous buffer systems. Another limitation is that the gels are cast in trays which are not covered. Because atmospheric oxygen has full access to the upper surface of the gel, acrylamide will not polymerize in this system. For agarose gels, the simplicity and ease of use of the horizontal system often make this system the best choice.

Casting a horizontal agarose gel

(A complete protocol is provided in Section 2.4.1) Horizontal gels are cast in trays which have removable ends. The ends are installed (often the ends are simply adhesive tape), and the molten agarose solution is poured into the tray. A comb is inserted so that the teeth penetrate the gel to within 1-2mm of the bottom of the tray. The overall gel thickness is generally 0.5-1cm. The gel is allowed to cool, the comb is removed, and the gel is mounted in the apparatus. Negative electrode Sample wells

Gel

Positive electrode

Negative electrode chamber

Sample wells

Applications

1.5.1 The Horizontal Gel System

Figure 1.5.1b shows a standard “mini”gel apparatus. Such gels, generally 10cm x 10cm or smaller, have become the standard for many applications, because of their ease of preparation and handling, and short run times. As with the “full size” system, the gels are cast between glass plates, but in the mini-gel system, the cassettes are mounted onto a “U” shaped frame, so that the cassettes themselves form the sides of the negative electrode chamber. This assembly is placed in a tank of buffer which contains the positive electrode. This means that the gels are effectively submerged in buffer during the run, providing optimal cooling.

Electrophoresis

A gel electrophoresis apparatus must allow the researcher to maintain a uniform electric field across the gel, provide cooling to prevent thermal artifacts, and allow access to the gel for sample loading and monitoring the run. Two types of apparatus are in common use: vertical and horizontal. Vertical gel systems are further subdivided into slab gels and tube gels. In general, agarose gels are run in the horizontal format, while acrylamide gels are run vertically.

gel. This allows precise and reproducible control of the voltage gradient. Because of the high resistance of the thin gel, the apparatus must have provisions for cooling. In the system shown, the front of the gel cassette is exposed to the air, while the back of the gel is held against a metal plate which dissipates heat rapidly. In some systems, the upper buffer chamber extends almost to the bottom of the gel, and the upper buffer is used for cooling. The relatively small amount of current carried through the gel means that buffer recirculation is generally not required.

Gel plates

Gel Negative electrode chamber

Cooling plate Retaining clip

Sample wells

Gels Positive electrode chamber

Gel plates

Retaining clip

Positive electrode chamber

Figure 1.5.2a The Vertical Gel Electrophoresis Apparatus.

Figure 1.5.2b The vertical mini-gel apparatus.

In general, vertical slab gels are loaded through the top, under a layer of buffer. The gels are monitored during the run through the front glass plate. The fact that the body of the gel in these systems cannot be accessed until the end of the run can be an inconvenience, and some sample recovery techniques used on horizontal gels are not available for vertical gels.

Casting a vertical slab gel

Figure 1.5.1a The Horizontal Gel Electrophoresis Apparatus. The gel rests on a platform which divides the apparatus into two chambers. Note that the buffer level is higher than the surface of the gel, so the two buffer chambers are connected. Samples loaded into the wells will migrate toward the positive electrode.

1.5.2 Vertical Gel Systems Slab gels

A typical vertical apparatus used for sequencing is shown in Figure 1.5.2a. This system shows the components common to all vertical slab systems. The gel is cast between two glass plates, separated by spacers, typically <2mm thick. The gel is mounted in the system so that the top is in contact with the negative electrode chamber, and the bottom is in contact with the positive electrode chamber. Unlike the horizontal system, the only connection between the buffer chambers is through the

Vertical gels are cast in a cassette made up of two glass plates separated by spacers which run along the sides of the plates. The bottom of the cassette is sealed by some temporary means (tape, agarose, or a gasket). The gel monomer solution is treated to initiate polymerization, and poured into the cassette. A comb is inserted into the top of the cassette to form the sample wells, and the gel is allowed sufficient time to polymerize. After polymerization, the bottom of the gel is unsealed, and the cassette is mounted in the apparatus.

Tube Gels

Tube gels were used frequently in the development of gel electrophoresis. Although they are still used for some applications (most notably for isoelectric focusing as part of 2D electrophoresis, Section 3.3.1), tube gels have been superseded by slab gels for most applications. Tube gels are cast (as the name implies) in glass tubes of 1-3mm diameter. The gels are run in a box which is divided into two chambers horizontally. The horizontal partition has gasketed mounting holes for the tube gels. The upper and lower chambers are filled with buffer, and current applied. The principle limitation of this system is that only one sample can be loaded per gel. USA: 1-800-526-3867 EUROPE: 441 482 646022

Applications

Electrophoresis

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Gel Electrophoresis of DNA and RNA

2.1 DENATURING POLYACRYLAMIDE GEL ELECTROPHORESIS OF DNA AND RNA

Overview / Sample Preparation / Gel Preparation / Run Conditions / Molecular Weight Determination / Manual Sequencing / Automated Sequencers / Differential Display / Genomic Analysis / RNA Mapping / DNA-Protein Interactions

2.2 NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS OF DNA

Overview / Sample Preparation / Gel Preparation / PCR Analysis / Mobility Shift Assay / DNA-RNA Purification from PAGE Gels

2.3 CONFORMATIONAL ANALYSIS

Heteroduplex Analysis / SSCP Analysis

2.4 AGAROSE ELECTROPHORESIS OF DNA AND RNA

Overview / Preparation of Agarose Gels / Restriction Digest Mapping / DNA-RNA Purification from Agarose Gels / In Gel Enzyme Reactions / Pulsed Field & Field Inversion Gel Electrophoresis (PFGE & FIGE) / RNA Electrophoresis

DNA Electrophoresis...Straight Up or with a Twist

he separation of nucleic acids by gel electrophoresis is theoretically straightforward. In aqueous solution, DNA and RNA derive their charge from the phosphate groups that occur with each nucleotide in their backbone. This fundamental structure does not vary with changes in base sequence. For this reason, nucleic acids possess an effectively constant charge to mass ratio. As such, their relative rates of migration in a sieving medium depend only upon molecular mass and conformational shape. In most cases, double stranded nucleic acids are rod shaped, and separation occurs on the basis of molecular mass alone, the exception being double stranded DNA with a mismatched base (or bases) leading to a bent rod conformation and altered electrophoretic mobility. In native conditions single stranded DNA or RNA folds into a variety of conformations, and such conformational differences can serve as the basis of electrophoretic separation as well. However, most electrophoresis of single stranded nucleic acids is performed under denaturing conditions, which disrupt the folded structure of these molecules, and lead to separation on the basis of molecular weight alone.

Electrophoresis of nucleic acids requires a gel matrix with a relatively open pore structure. Nucleic acids are generally larger than proteins. The RNA which codes an average sized protein (50,000 daltons or 450 amino acids) must contain 1350 nucleotides and possess a molecular weight of over 400,000 daltons. The gene for this protein, including introns, would extend into millions of daltons in size. DNA or RNA molecules containing less than one thousand bases are best resolved on polyacrylamide gels because of the superior resolution of this medium. Polyacrylamide can separate fragments differing by a single base pair. The uniform, small pore sizes available in polyacrylamide gels allow for the resolution of bands which differ in size by as little as 0.1% (1 base in over 1000). By far the most common use of Polyacrylamide Gel Electrophoresis (PAGE) of nucleic acids has been the sequencing of DNA, in which single stranded DNA molecules are separated under denaturing conditions. PAGE is also widely used under native conditions, to analyze PCR products or restriction digest products under 1000 bases in length. For the larger fragments which will not enter a polyacrylamide matrix, agarose must be used. Agarose gels have the capacity to fractionate DNA or RNA molecules millions of bases in size. While PAGE offers the advantages of fast runs, high resolution, and rapid staining of thin gels, as well as the thermal stability required by denaturing conditions, the utility of PAGE is limited in situations where large molecules must be run or samples must be recovered after electrophoresis. In these cases, agarose is the matrix of choice. 44

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Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

2.1 Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA

Applications

Both formamide and urea effectively lower the melting point of the DNA molecules, allowing the structures to fall apart at lower temperatures. Generally, concentrations of urea or formamide are chosen to give melting temperatures around 50 °C, and gels are run at that temperature. RNA is often denatured with harsher agents, because RNA tends to form stronger structures. RNA denaturation is discussed in section 2.4.6 on RNA electrophoresis.

Denaturing gels for sequencing are poured between two glass plates separated by spacers. The spacers are typically no more than 0.2mm in thickness. The extreme thinness of the gel allows air bubbles to be trapped in the gel during pouring. Such bubbles are very hard if not impossible to remove. Because oxygen inhibits the polymerization process, even a small bubble can create a hole in the gel which is large enough to prevent the use of several lanes. Bubbles can be avoided by using scrupulously clean plates, by siliconizing one plate, and by vigilance during the casting process.

Electrophoresis

The electrophoretic analysis of single stranded nucleic acids is complicated by the secondary structures assumed by these molecules. Separation on the basis of molecular weight requires the inclusion of denaturing agents which unfold the DNA or RNA strands and remove the influence of shape on their mobility. Nucleic acids form structures stabilized by hydrogen bonds between bases. Denaturing requires disrupting these hydrogen bonds. The most commonly used DNA denaturants are urea and formamide. Each of these forms hydrogen bonds with the DNA bases, “saturating” H-bond sites and preventing the formation of inter-base bonds (Figure 2.1.a).

2.1.2 Gel Preparation

Protocol 2.1.2a Denaturing Gel Preparation Using National Diagnostics’ UreaGel 6 1. Clean glass plates thoroughly. Use a laboratory detergent, and scrub with a gloved hand or a paper towel. Rinse thoroughly with tap water; observe the flow of water over the plate (Unclean areas often cause distortions in the flow). Wipe the plate with a glass cleaner and rinse thoroughly with distilled water. Rinse with ethanol and wipe dry. 2. Apply Glass Free™ to one glass plate to ensure the gel will release from one plate after electrophoresis. This also will reduce the tendency of the treated plate to trap air bubbles. 3. Assemble the cassette. Use the clamps provided with the apparatus, if available. If clamps are not provided, use binder clips, one every 20 cm along both edges. Place the cassette on a level surface which is shorter than the plates, so that the top and bottom of the gel extend beyond the surface. A thick book works well. 4. Add 80ml of UreaGel 6 Monomer Concentrate and 20ml UreaGel Complete Buffer to a thick-walled Erlenmeyer flask.

Figure 2.1a The denaturation of DNA by urea.

2.1.1 Sample Preparation Denaturing DNA samples

DNA samples for denaturing gel electrophoresis must be denatured prior to loading to avoid time dependent denaturation artifacts on the gel. This is usually carried out by diluting the sample into 95% formamide and heating to 95°C (see Protocol 2.1.5c on DNA sequencing for a formula for the loading buffer). Loading the proper amount of DNA is critical for good results. Too little DNA will not be detected, while overloading lanes leads to smearing of bands. Acrylamide gels have a relatively high sample capacity—up to 10 µg can be loaded per lane in many cases.

Determining sample concentration

The concentration of DNA in the sample may be determined in several ways. The most straightforward is to make use of the absorbance at 260nm of the nucleotide bases. Pure DNA at a concentration of 50 µg/ ml has an A260 of 1.0 (Concentration is linear with absorbance by Beer’s Law). The purity of the DNA may be checked at the same time: pure DNA has a ratio of A260/ A280 of 1.8. A lower ratio indicates protein contamination; a higher ratio indicates substantial RNA content. Lower concentrations of DNA may be assayed by taking advantage of the fact that the fluorescence of DNA/ethidium bromide complexes is proportional to the concentration of DNA in the sample. Levels of DNA as low as 10 ng can be quantified in a 10µl volume by diluting the DNA into buffer or water containing 0.5µg/ml ethidium bromide. CAUTION: ETHIDIUM BROMIDE IS A POWERFUL MUTAGEN. Comparing the fluorescence of serial dilutions of sample with the fluorescence of known standards allows the determination of the DNA concentration in the original sample.

5. If desired the solution may be degassed for two minutes. Apply a vacuum (an aspirator is sufficient) while stirring or shaking. Bring to room temperature before polymerization. 6. Add 800 µl FRESHLY PREPARED 10% Ammonium Persulfate , swirl gently to mix, and cast the gel. TO CAST: with the gel lying flat, pour gel solution into the gap between the short and long plates, along the entire width of the gel. Capillary action will draw the solution into the cassette. Pour solution steadily to keep the top gap filled, and tilt the cassette 10-20° to encourage flow toward the bottom. Tap the cassette with a rubber stopper rapidly to keep bubbles from being trapped. If the plates are clean, the solution will flow from top to bottom smoothly in a line across the cassette. When the solution has reached the end of the cassette, return the gel to a level position, and insert the comb, flat edge first. Allow the gel to polymerize one to two hours.

NOTE: After two hours of polymerization wrap each end of the gel cassette with clear plastic wrap. This is important to keep the ends of the gel from drying and to maintain sample well integrity. Appropriately wrapped gels may be stored for up to 48 hours at room temperature.

7. Mount the gel in the running apparatus. Fill the upper chamber with buffer and remove the gel comb. Replace the comb with the teeth facing the gel, and insert until the teeth just penetrate the gel (no deeper than 1mm). Fill the lower chamber. 8. Prerun the gel for 15-30 minutes before loading the samples, or as recommended for the apparatus used. For small (35ml, 20X40X0.02cm) gels use 30-35 Watts. For large (70ml, 40X40X0.02cm) gels use 55-65 Watts. The gel temperature should be between 45-50°C.

Ready-to-Use Gel System for 6% Denaturing Gels UreaGel-6 EC-836 Convenient system for casting 19:1 6% denaturing gels with Acrylamide, Bis, TEMED, TBE and urea. (pg. 12)

TBE 10X EC-860 Formulated with 18 MegOhm water. 0.2 micron filtration. Costs the same as bench-made buffers. (pg. 20)

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Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

2.1.3 Run Conditions

Applications

Electrophoresis

Temperature

The most critical parameter in denaturing DNA-PAGE is gel temperature. Highly concentrated urea (6-7M) is the most commonly used denaturant, but to be fully effective the temperature must be maintained above 40°C. Denaturing PAGE gels are generally run with the temperature in the range of 45-60°C, which is maintained by running the gel at constant power (wattage), as opposed to constant voltage or current. Because power measures the energy transferred through the gel (Section 1.2.2) maintaining constant power provides constant heat output and thus a stable temperature. It is crucial that denaturing gels be pre-run for at least 30 minutes prior to loading to bring the gel up to operating temperature.

Denaturants

Some nucleic acid molecules are particularly difficult to denature. Sequences with a high guanine and cytosine content have more hydrogen bonds than adenine and thymine rich sequences (Section 1.1.1) and require more vigorous denaturation. Sequences which can fold back on themselves (Section 1.1.1) are also difficult to denature fully, because the two complementary sequences are “tethered,” increasing the likelihood of their re-annealing after denaturing. Difficult samples such as these may require gel temperatures in the 60-80°C range, which can give fuzzy bands, distorted gels and may lead to cracked plates. Alternatively, formamide—a more active denaturant—may be included in the gel at concentrations up to 40%. This strategy is often used in DNA sequencing to alleviate “GC compressions”; regions of poor resolution caused by high GC content regions in the sample.

Buffer

Another important consideration in running denaturing PAGE is buffer selection. Some buffers used in molecular biology, most notably Tris-acetate EDTA (TAE), are easily exhausted. This means that they lose buffering capacity during the run, resulting in pH shifts at the ends of the gel. Tris-borate EDTA (TBE) is the buffer of choice for denaturing PAGE of nucleic acids. It has a high buffering capacity and can be run at high voltage for hours without exhaustion. Its only drawbacks are that it can interfere with some DNA recovery protocols, and that it forms complexes with glycerol which can distort gel patterns. If glycerol is required as part of a sample preparation, Tris-taurine EDTA (TTE) buffer is recommended. TTE has buffering capabilities similar to TBE, but shows no artifacts in the presence of glycerol.

Applications of Denaturing PAGE of DNA and RNA 2.1.4 Molecular Weight Determination Molecular weight determination is the most basic use of denaturing polyacrylamide gel electrophoresis. Samples are run versus standards of known molecular weight, and a calibration curve of relative mobility (or distance migrated) versus the logarithm of the size is established. Size can be expressed as molecular weight or number of bases. It is important to bear in mind that sequences of identical length may vary in GC content, and therefore in weight. Additionally, strong base pairing, hairpins and other residual secondary structure may perturb the mobility of a given fragment. Accurate molecular weight determination is best undertaken using size standards which are derived from the same DNA as the sample such as overlapping PCR products, cleavage fragments or synthetic products.

2.1.5 Manual Sequencing Overview

In traditional gel-based sequencing, DNA sequences are determined by a two step process. In the first step the sample DNA is used, either directly or as a template, to generate sets of fragments. Each set contains multiple lengths of DNA, all of which end in one (or sometimes two) of the four nucleotide bases. These fragments are generally radiolabeled to facilitate detection. In the second step, the fragments are separated on a denaturing PAGE gel. Each band on the gel represents a position in the DNA sequence, and each position appears only in the fragment set which terminates in the correct base(s). Autoradiography is performed on the gel to visualize the bands. Figure 2.1.5a shows the generation of fragments by two methods, and the gel pattern which results from electrophoretic separation of these fragments.

Buffer Gradients

Gradients of buffer concentration can be employed to compress regions of the gel, thus providing more information per run. Fragment mobility is proportional to the logarithm of the molecular weight, so for 10 base fragments which differ by one base in length, the difference in mobility is 4%. For a one base difference at 100 bases, it is 0.3%, and at 500 bases, one base difference gives only a 0.03% difference. Thus, at the bottom of a 40cm gel, a one base difference in length will give band separations of 1-2cm, while 100 base fragments, having migrated 20cm, will separate from 101 base fragments by only 0.1cm. Clearly, compressing the pattern at the bottom of the gel, so as to allow longer runs without losing the smaller bands, will allow greater resolution of the larger fragments in the sample. This is accomplished by lowering the resistance across the lower portion of the gel which leads to a lessening of the voltage drop across this region. In the lower voltage gradient, bands will migrate more slowly. Small bands will thus migrate rapidly through the upper portion of the gel, and then slow down as they approach the end. This allows longer runs, which facilitates resolution of the larger bands on the gel. The drop in resistance at the bottom of the gel can be accomplished by use of wedge spacers, which are wider at the bottom, increasing the cross sectional area of the gel. More often, however, a buffer gradient is used, with a higher conductivity buffer in the lower buffer chamber. (Protocols are given in Section 2.1.5, Protocol 2.1.5d, DNA sequencing) 46

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Maxam and Gilbert Sequencing

Sanger Sequencing

Figure 2.1.5a The two methods of conducting sequencing analysis. The Maxam and Gilbert method employs a set of cleavage reactions to generate the necessary fragments while the Sanger method employs a polymerase.

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Maxam & Gilbert Sequencing

There are four chemical cleavage reactions at the core of the Maxam and Gilbert sequencing system. Figure 2.1.5c shows an example from these reactions, the reaction cleaving specifically at guanine. The other three reactions cleave at G+A, C+T, or C. Guanine and cytosine, therefore, give bands in two lanes, adenine and thymine in only one. An example of the gel pattern produced is presented in Figure 2.1.5b. The DNA to be sequenced must first be end labeled, at one end only. This is accomplished by kinase treatment with 32P ATP, which labels both ends, followed by restriction digestion and isolation of the two labeled fragments (Protocol 2.1.10a). Alternatively, digestion of a plasmid containing a clone of the DNA of interest with an appropriate enzyme can yield a unique labeling site. Plasmid vectors containing the rare site for Tth111I, which leaves a single 5’ base overlap, have been generated for this purpose. Cleavage with Tth111I leaves a G at one end and a C at the other in these vectors. By filling in the gap with Klenow polymerase fragment in the presence of dGTP or dCTP, one end or the other can be labeled specifically. Labeled DNA is first precipitated to remove any salts which might interfere in the cleavage reactions. It is then modified, cleaved and run on a denaturing gel for analysis. NB: THE HYDRAZINE AND DMS USED IN THESE PROTOCOLS ARE TOXIC AND VOLATILE. KEEP TUBES SEALED AND WORK IN A HOOD.

Maxam and Gilbert Sequencing Reactions 1. Precipitate the substrate: To the 32P labeled DNA, add 0.1 vol. 3M sodium acetate and 1 vol. Isopropanol. Precipitate at -70°C for 10 minutes, and centrifuge at max RPM in a microcentrifuge for 5 minutes to collect the DNA. Wash the pellet twice with 1 ml cold 70% ethanol to remove all salt. Redissolve the DNA in 45 µl of sterile water. Count one microliter of the solution in scintillation cocktail to confirm >5x103 cpm total counts. 2. Aliquot 10µl of the DNA solution into each of 4 tubes. Label the tubes C, G, C+T, G+A. 3. Reactions: C: Add 10µl 2.5M NaCl and mix well. Add 30µl of hydrazine (toxic!) and incubate at 25°C for 7-9 minutes. G: Add 200µl of: 50mM sodium cacodylate, pH 8, 1mM EDTA. Mix well and add 1µl dimethyl sulfate (DMS) (Toxic!) and incubate at 25°C for 4-5 minutes. C+T: Add 10µl H2O and mix well. Add 30µl hydrazine and incubate at 25°C for 7-9 minutes. G+A: Add 25µl of formic acid, mix well and incubate at 25°C for 4-5 minutes.

Applications

Sanger dideoxy terminator sequencing is more commonly used than the Maxam and Gilbert reactions. Although it requires prior knowledge of at least 15-20 bases of the sample sequence, it is far less laborious and more reliable, particularly for long substrate sequences. In this system, the sample DNA is used as a template for a DNA polymerase, typically a bacteriophage polymerase (T4 or T7). Four polymerase reactions are set up for each substrate, each containing enzyme, primer and sample DNA, along with dNTP’s. In addition, each reaction contains one of the four dideoxy NTP’s. Dideoxy nucleotides do not have a 3’ OH. They are linked to the growing DNA chain through their 5’ OH, and the chain stops there for lack of a 3’ OH to link to the next base. Each reaction contains one of the four bases as a dideoxy NTP, thus each reaction will contain only fragments which terminate at that base. Proper balance of the levels of DNA, primer, enzyme, ddNTP and dNTP allow reactions which can give readable sequence out to 1500 bases from the primer. In most dideoxy sequencing, 35S labeled dNTP(s) are included in the polymerase reactions to label the fragments produced. Alternatively, 33P dNTP’s, 32P labeled primers, or fluorescent labelling can be used.

Protocol 2.1.5a

Electrophoresis

Of the two methods used to generate the fragments for sequencing gel analysis, the Maxam & Gilbert chemical cleavage method was the first to be widely used. Although no longer predominating, Maxam & Gilbert sequencing possesses significant advantages in certain applications. The four sets of reactions involved in this method cleave the DNA at specific bases or base sets to produce four sets of fragments. No prior knowledge of the DNA sequence is required as the sample DNA is itself processed into fragments. Maxam & Gilbert chemistry is thus useful for analyzing fragments of completely unknown sequence and is essential for footprinting protocols (Section 2.1.9).

4. Stop the reactions:

Stop buffers: G reaction: Add 50µl of:1.5M sodium acetate pH 7, 1M mercaptoethanol, 100µg/ml tRNA. All other reactions: Add 200µl of 0.3M sodium acetate, pH 7, 0.1mM EDTA, 25µg/ml tRNA.

Ethanol precipitation: Add 750µl of Ethanol, and transfer reactions to a -70°C bath for 5 minutes. Collect DNA by microcentrifugation for 5 minutes. Discard the supernatants as appropriate for DMS or hydrazine waste. Rinse the pellets twice with 70% ethanol. Redissolve the pellets in 300µl of water, add 30µl of 3M sodium acetate and 1ml of ethanol. Pellet DNA and wash twice with 70% ethanol. Allow the pellets to air dry.

5. Piperidine cleavage reactions: Resuspend pellets in 75µl of 10% piperidine, and transfer to screw top tubes. It is essential that the tubes used for the piperidine reaction seal well in order to ensure that the reaction goes to completion. Incubate the tubes at 90°C for 30 minutes. Cool the tubes, centrifuge briefly to collect the condensate, and evaporate to dryness in a speedvac. Redissolve the pellet in 40µl of water and dry again. Repeat the rehydration and drying once more to ensure that all of the piperidine has been removed. The samples are now ready for denaturing PAGE. (Sections 2.1.2, 2.1.3, 2.1.5).

DMS

Figure 2.1.5b In a Maxam and Gilbert gel, the identity of guanine or cytosine in the sequence can be assigned most easily because two of the four reaction sets cleave at those bases alone. Adenine or thymine are slightly more difficult, being represented by those bands in the G+A or C+T lanes which do not appear, respectively, in the G or C lanes.

Piperidine

Figure 2.1.5c Maxam and Gilbert DNA sequencing reaction specific for Guanidine residues. The Guanine base is first modified with Dimethyl Sulfate (DMS), which makes the chain susceptible to cleavage by piperidine, destroying the Guanidine residue and releasing a labeled fragment for electrophoresis. USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Applications

Electrophoresis

Sequencing - Sanger Method

In Sanger dideoxy terminator sequencing, the sample DNA is used as a template for a DNA polymerase. Four polymerase reactions are carried out involving enzyme, primer and sample DNA, along with dNTP’s. Each reaction also contains one of the four dideoxy NTP’s. When a dideoxy NTP is added, chain lengthening terminates because ddNTP nucleotides lack 3’ hydroxyl groups by which to form the next phosphodiester bond. Each reaction contains one of the four bases as a dideoxy NTP, thus each reaction results in fragments terminating at that base. The four reactions produce four collections of fragments with lengths reflecting the sequence positions of each of the four respective bases. Numerous commercial kits are available for Sanger sequencing. These kits provide excellent and consistent results without the need for the researcher to titrate dideoxy mixtures for maximum efficiency. An outline of the method is presented here. However, it is strongly recommended that the user follow the protocol provided with their particular kit. For optimum performance, dideoxy sequencing requires a single stranded substrate. The most prevalent artifact associated with dideoxy sequencing is the appearance of BAFL’s (Bands Across Four Lanes), which are due to polymerase “pause” sites, at which the enzyme tends to fall off of the DNA, resulting in a non-dideoxy termination. This effect is exaggerated when the polymerase is required to process double stranded DNA. Single stranded substrates are generally prepared by cloning the DNA of interest into vectors derived from the bacteriophage M-13. Phage M-13 replicates its DNA in the bacterium in double stranded form, allowing easy cloning manipulations, but its phage form contains circular single stranded DNA. Protocol 2.1.5b Purifying SS M-13 DNA

Protocol 2.1.5c Dideoxy Sequencing (Taq Polymerase) 1. Anneal the primer:

Dissolve 0.5pmoles of M-13 substrate in 10µl of Taq buffer: 50mM Tris HCl, pH 9.0, 15mM MgCl2. Add 0.5pmole of primer in 1µl of water or TE buffer. Incubate at 65°C for 10 minutes, then allow to cool to room temperature over 30 minutes. This is most easily accomplished by placing the tube in a 100ml beaker of water at 65°C and allowing the beaker to cool on the benchtop.

2. Set up the termination reactions:

Aliquot 2.5µl of A,C,G and T termination mixtures into appropriately labeled tubes. The termination mixtures contain 8µM of the dNTP targeted for termination, 800µM each the other 3 dNTP’s, and 0.1mM EDTA. In addition, each mixture contains one ddNTP, as follows: A: 125µM ddATP C: 50µM ddCTP G: 20µM ddGTP T: 100µM ddTTP 3. Labeling reaction (initial primer extension):

Add 2µl of a mixture of 1.6µM each of all 4 dNTP’s, 1µl 10mCi/ml S-dATP, and 2 Units of Taq polymerase (diluted in Taq buffer to 2U/µl). Place in a 40-45°C water bath for 3-5 minutes. Do not start the reaction at 45°- this may denature some primer-template complexes.

4. Termination:

Add 3.5µl of labeling reaction to each termination tube. Mix well and incubate at 60°C for 5-10 minutes. Add 4µl of loading dye (95% Formamide, 20mM EDTA, 0.05% each Bromophenol Blue and Xylene Cyanole), and heat to 95°C for 2 minutes prior to loading on a denaturing PAGE gel.

1. Precipitate the phage with Polyethylene Glycol (PEG):

Remove bacteria from 1.5 ml of culture by centrifuging at 12 - 14 K RPM in a microcentrifuge. Transfer the supernatant to a fresh tube, containing 0.2 ml 2.5M NaCl + 20% PEG 8000 and mix well. Incubate 15 minutes at room temperature. Pellet the phage particles in a microcentrifuge at 14K RPM 15 minutes at 4°C. Remove supernatant with a pipette, being careful not to disturb the very small phage pellet. Briefly centrifuge the tube to bring residual supernatant to the bottom and remove it with a pipette.

2. Purify the DNA:

Vortex the pellet in 50µl T10E1 buffer. Add 50µl phenol equilibrated with Tris pH 8.0. Vortex 30 seconds - 1 minute. Separate the phases in a microcentrifuge at 14K RPM for 2 minutes. Recover the upper phase, being careful not to disturb the interface layer. (Note: leaving some of the supernatant over the phenol phase ensures a cleaner preparation.) Add 300µl Ethanol and 15µl 3M sodium acetate, pH 5.2. Mix well and incubate at room temperature for 15 minutes. Collect the DNA in a microcentrifuge at 14K RPM 20 minutes at 4°C. Remove the supernatant and wash the pellet twice with 70% ethanol. Proceed with primer annealing: 1.5 ml of culture should yield > 5µg of DNA, sufficient for 1-2 sequencing reactions. Figure 2.1.5d In Sanger sequencing four reactions are run, each designed to terminate the growing DNA chain at one of the four bases (the G reaction is shown in detail). The result is four collections of fragments whose comparative lengths indicate the positions of the four bases (the sequence) of the DNA under study.

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Gel Electrophoresis for DNA Sequencing

Denaturing PAGE gels for DNA sequencing generally employ 6-8 M urea as denaturant and TBE as buffer system. They are poured as described in Section 2.1.2. After a 2 - 2.5 hour run, a 6% polyacrylamide sequencing gel will give 200-250 bases of readable sequence starting at or close to the end of the primer. A number of variations, enhancements and improvements to the basic PAGE gel have been developed to increase the number of bases which can be read from one gel. The pattern of sample loading can be important. The best method is to load in a pattern such as GATCGTAC, in which each reaction is loaded twice, and each reaction borders on every other reaction once. At the top of the gel, where bands are compressed, this allows unambiguous assignment of order and thus base position. Loading in this pattern can extend readability by up to 50 bases, while at the same time diminishing errors in reading throughout the sequence. Significantly more sequence information can be derived from a set of reactions by “double loading”. Portions of the samples are loaded into half the wells in the gel, and the gel is run for 1-2 hours. The power is turned off, and the remaining portions of the samples are loaded into the remaining lanes. Running the gel for 1-2 more hours gives runs of 2 and 4 hours, which will allow sequence to be read from the primer out to 350-400 bases. The use of a wedge gel or a buffer gradient system can extend read length by up to 30%. Wedge gels, cast with spacers wider at the bottom of the gel, work well but are inconvenient to dry. Buffer gradient gels are more difficult to pour, but easier to handle after the run. Both options work by decreasing the electrical resistance in the bottom portion of the gel. Wedge gels have a wider cross sectional area at the bottom, while buffer gradient gels have a higher salt content in that area. The decreased resistance causes the voltage drop across the lower portion of the gel to be diminished. The DNA molecules in this region are subjected to less

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Pouring a Buffer Gradient Denaturing Gel 1. Prepare 2 gel solutions, containing the desired concentration of Acrylamide/Bis-Acrylamide and Urea. One solution should contain TBE at 0.5X, and one at 2.5X (containing 10% sucrose). Add Bromophenol Blue to the 2.5X TBE solution to 0.001% (just sufficient to give a visibly blue tint). The volume of each solution should be 75% of the amount needed to completely fill the gel cassette. 2. Add APS and TEMED to the two solutions to initiate polymerization. 3. In a 25ml pipette, draw up first 12.5 ml of 0.5X TBE gel solution, then 12.5ml of Blue 2.5X TBE gel solution, for a total of 25ml. 4. Draw 2 - 3 air bubbles through the pipette, to mix the two solutions at the interface. 5. Fill the cassette with the solution in the pipette. Fill the remainder of the cassette with the 0.5X TBE gel solution. 6. Allow to polymerize 2 hours, and run with 0.5X TBE in the upper buffer chamber, and 1X TBE in the lower chamber.

In some situations, the information read from a gel is of sufficient length, but insufficient quality. Substrates with a high G-C content will tend to retain enough secondary structure, even in 6M Urea at 55°C, to cause anomalous gel migrations. On the gel, this is observed as multiple bands in G or C lanes that are too close together for accurate reading. These regions are known as G-C compressions, and they can be very hard to resolve. Inclusion of a stronger denaturant in the gel alleviates most, if not all, GC compression problems. The denaturant of choice is formamide, which is strongly denaturing to DNA, uncharged, and easily miscible with high concentrations of urea in water. Gels with 6M urea and 40% formamide are typically formulated to resolve GC compressions. Formamide solutions must be made fresh, using deionized formamide. Another source of difficulty in reading sequencing gels is the inclusion of glycerol in samples run on gels containing TBE. Glycerol, often used to stabilize the polymerase in the sequencing reactions, forms a complex with the borate in TBE. This creates a “salt wave” of altered conductivity, which migrates through the gel with the DNA, distorting a narrow range of DNA sizes. This problem is alleviated by using non-borate buffers, such as TTE, in gels used to run glycerol containing samples.

Products for Manual and Automated Sequencing UreaGel-6 EC-836 Convenient system for casting 19:1 6% denaturing gels. Contains acrylamide, bis, TEMED, TBE and urea. (pg. 12)

Automated sequencing systems make use of fluorescent dye labeling, in combination with laser scanning and computerized data acquisition and processing to carry out the electrophoresis of up to 96 sequencing reactions on a single gel, and read over 1,000 bases from each reaction. A single run on an automated sequencer can thus produce as much data as 40 manual gels. Reactions for automated sequencing use the same Sanger dideoxy chemistry as manual sequencing systems. The key difference lies in how the fragments are labeled for later detection. Instead of radiolabeling, highly fluorescent dye molecules are linked either to the primer (dye-primer) or the dideoxy NTP’s (dye terminator). Excess dye is removed in a post-reaction cleanup step, and the products are separated on a denaturing polyacrylamide gel as used for manual sequencing. The gel is run while mounted on a detector, which constantly scans a laser across all lanes at the bottom of the gel. When a labeled band crosses this detection zone, its fluorescence is detected and recorded. The pattern of fluorescent flashes over time is interpreted into a DNA sequence using a computer.

Applications

Protocol 2.1.5d

2.1.6 Automated Sequencers

Electrophoresis

force, so they slow down relative to bands in the upper portion of the gel. The net effect is to “compress” the bands as they migrate into the bottom of the gel, allowing longer runs and more readable sequence per gel. A similar, although less marked effect may be obtained simply by filling the upper chamber with 0.5X TBE, and the lower with 2X TBE. The pre-run gradient will not be as continuous as one poured into the gel, and some information may be lost at particularly steep points in the gradient.

One of the strengths of this system is that multiple colors of fluorescent dye are available (note that many of these dyes are fluorescent in the UV or IR regions, so the term “color” is used as a shorthand for “fluorescent at a different wavelength”. In general, a different color is used for each ddNTP reaction. As a result, all 4 reactions can be run on a single lane. Each band is identified with a position in the DNA by its elution time, and with a base by its color. Although automated sequencing uses unique labeling and processing techniques, the electrophoretic separation at its core is essentially the same as that for manual sequencing. The key differences are in the much higher standard of purity required, and the focus on larger DNA fragments. Automated runs may take as long as 12 hours to complete. The gel must remain intact and unaltered during that time, at elevated temperatures. Additionally, the use of laser scanning means that ultra low levels of contaminant will show up as elevated background fluorescence. Also, gel results are processed according to parameters set in the computer software, which must be reproducible from gel to gel. All of this demands that the acrylamide, urea and buffers used be of exceptionally high purity.

Figure 2.1.6a An automated DNA sequence produced on an ABI 377 equipped with 36 cm WTR plates running at 2X speed for 7 hours.

For most automated sequencers, the best matrices are modifications of the traditional 19:1 manual sequencing gels. The need for modification arises because, while manual sequencing generally aims to read 400 bases at most from a reaction, automated sequencing in some machines allows over 1000 bases to be read. In these machines, a more open pore structure is required to allow the resolution of the larger fragments.

SequaGel XR EC-842 SequaGel XR gives the longest reads of any matrix on the Li-Cor 4000 and 4200 sequencers. (pg. 14)

TBE 10X EC-860 Formulated with 18 MegOhm water. 0.2 micron filtration. Costs less than bench-top buffers. (pg. 20)

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Applications

Electrophoresis

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

2.1.7 Differential Display

2.1.8 Genomic Analysis

Differential display is a variation of standard PCR, allowing the amplification of a large population of fragments, rather than the specific amplification of one band. Specificity is reduced in two ways. First, random primers are used at one end of the amplification. A mixture of all possible hexamers (46=4096 primers) is used. This allows many different molecules to be amplified.

RAPD, the more simple of the two techniques, uses PCR primers which are substantially shorter than the usual 17-21 bases. These short (8-12 base) primers will anneal to multiple sites on the DNA sample. When these annealing sites are within 1000 bases, and properly oriented (figure 2.1.8a), amplification occurs. Provided that the amplification conditions are well controlled, the population of fragments generated will reflect the source of the substrate DNA, and analysis on a denaturing polyacrylamide gel will provide a fingerprint for that individual genotype.

Differential display is a powerful technique for detecting and quantitating changes in gene expression patterns between differently treated cells. Fragments of those genes which are induced or suppressed can be identified and isolated for further analysis, with no prior knowledge of the sequences involved. The technique is PCR based, and yields results in only 1 - 3 days.

The second drop in specificity is provided by using the poly A tail present on the vast majority of RNA species. A poly dT primer is used to prime on this tail. The 3’ end of the poly dT primer has a pair of non T bases, which is sufficient to “anchor” the primer to the end of the coding sequence (Figure 2.1.7a). There are thus 32 = 9 different possible primers at this end. Amplifications run with random hexamers and the poly dT primers on cDNA (that is, DNA copied from mRNA) taken from treated and untreated cells, gives a pattern of bands, each of which represents an amplified fragment of an expressed gene. A fragment which appears in one sample but not the other is either induced or suppressed by the treatment. Such bands may be cut out and re-amplified (using the same primer set as in the original reaction) for further analysis including sequencing, cloning, probe synthesis, etc. Differential display is covered by patents owned by GenHunter. GenHunter recommends the use of UreaGel-6 for differential display analysis: GenHunter Corporation - 624 Grassmere Park Dr. - Nashville, TN 37211 - (800) 311-8260 - info@genhunter.com.

mRNA Pool

Denaturing Polyacrylamide gel electrophoresis, coupled with variations of PCR amplification, provides a powerful set of tools for “fingerprinting” of genomic DNA. Two popular techniques are Random Amplification of Polymorphic DNA (RAPD), and Amplified Fragment Length Polymorphism (AFLP). In both of these techniques, nonspecific PCR is used to emphasize differences between genomic DNA samples.

AFLP, while more involved, has been shown in some cases to be more reliable and to generate more useful data than RAPD. These advantages arise because AFLP combines a more specific PCR system with restriction analysis, giving better control over the size and identity of the fragments generated. To carry out AFLP analysis, the researcher first digests the genomic DNA with two restriction enzymes. Generally, one “six-cutter” and one “four-cutter” are used, to generate a maximum number of optimally sized fragments. After digestion, 20-40 base adapters are ligated onto the ends of the digested fragments. These adapters allow the use of 17-20mer specific primers in the amplification step, which greatly enhances the reproducibility of the technique. The ligation reaction is then diluted and used as a substrate for PCR amplification. The amplification step of AFLP uses primers which contain 14-18 bases designed to anneal to the ligated adapters. These primers also contain 2-4 additional bases at their 3’ ends. These additional bases must match the sequence of a ligated genomic restriction fragment if that fragment is to be amplified. The number of 3’ additional bases will thus help determine the number of fragments observed after AFLP amplification (longer extensions will find fewer matching fragments), and the choice of those bases will determine the pattern of fragments seen. Denaturing gel electrophoresis will provide a readily analyzed pattern of amplified fragments. Given that a library of primers is possible for any single set of adapters, a very specific fingerprint can be developed for any genomic sample.

Reverse Transcription

AFLP Primer

Amplification Adapter

Restriction Site

Selective Bases Complementary

Amplification No Amplification Figure 2.1.7a Differential display is a PCR based technique that generates a characteristic set of DNA fragments from the messenger RNA pool within a cell. The use of random hexamers (brown) in combination with oligo-dT primers (blue) allows the amplification of a population of DNA species which changes with the composition of the starting RNA pool. Differential display is a powerful tool for the analysis of gene expression.

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Selective Bases Not Complementary

Figure 2.1.8a AFLP is a dependable, robust means of genetic fingerprinting. Genotypic variations detected by this technique include the location of restriction sites and also polymorphisms adjacent to these restriction sites. The amplified fragments are electrophoresed on a denaturing PAGE gel to yield the AFLP fingerprint.

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

2.1.9 RNA Mapping

Protocol 2.1.9a S-1 Mapping NB: ALL REAGENTS AND GLASSWARE MUST BE RNase FREE OR DEPC TREATED. 1. Probe preparation: S1 mapping requires a probe which is labeled at only one end. This can be accomplished in a number of ways, depending upon the available starting materials. Asymmetric restriction digestion of a cloned substrate can be manipulated to produce only one recessed 3’ end, which can be extended by Klenow fragment in the presence of labeled dNTP’s. Alternatively, an end labeled oligonucleotide may be extended with Klenow fragment, and the duplex cleaved with a restriction enzyme to produce the required probe. However the probe is produced, it is generally purified on an alkaline agarose gel (Section 2.4.1). Use a 1.2% low-melting gel. Locate the labeled band by autoradiography (Section 4.1.2), excise and trim it with the aid of a Geiger counter to remove excess agarose.

Melt the excised band at 65°C, and add an equal volume of TE buffer. Add 10µg of tRNA, extract with 2x1volume of phenol (Tris equilibrated to pH 8). Ethanol precipitate the supernatant with 0.1 vol of 3M sodium acetate and 2 volumes of ethanol. Redissolve the pellet in 100µl of 0.3M sodium acetate.

S1 Mapping

Nuclease S1 will digest only ssDNA or ssRNA. If a duplex of DNA and/or RNA strands has single stranded overhangs or unhybridized internal loops, these will be digested away. The remaining intact nucleic acid fragments represent regions of identity between two strands of the duplex. If one of the strands is labeled at one end, the length of labeled fragment remaining after hybridization and nuclease digestion reflects the point on the probe where the two sequences diverge. This is the basis for S1 mapping of transcriptional start sites (Figure 2.1.9a). A probe is chosen that is complementary to the RNA, and extends past the anticipated start site. The 5’ end is 32P labeled, and chosen to fall within the coding region of the mRNA, so that it will be protected from digestion. After hybridization, the 3’ overhang of the probe is digested away, and the size of the remainder of the probe is accurately determined on a denaturing PAGE Gel. The distance between the known labeling site and the new end of the probe gives the transcriptional start site to within 1 base. Labeled Probe

Applications

Electrophoresis

In studies of transcriptional regulation, it is often necessary to determine the structure and/or amount of a given RNA species. Several techniques have been developed making use of the fact that some nucleases will only digest single stranded DNA or RNA. Duplexes are resistant to digestion. By hybridizing samples with labeled single stranded probes, the amount of probe complementary material in the sample mixture can be assayed, as well as the probe sequence involved. Such assays can be carried out on complex mixtures of RNAs, as the labeled probe will only hybridize to the target RNA sequence. The most common uses for these techniques are to find transcription start sites and splice sites, and to quantitate specific RNAs.

2. Hybridization:

Add 1x105 counts of labeled probe to 20-25µg of RNA at 4°C. Add sufficient 0.3M sodium acetate to bring the volume to 100µl. Add 300µl of ethanol, pellet and wash the precipitated DNA with 70% ethanol. Allow the pellet to air dry. Redissolve the pellet in 20µl of S1 buffer: 80% formamide, 40mM PIPES pH6.5, 400mM NaCl, 1mM EDTA. Heat to 65°C for 10 minutes and let stand overnight at room temperature.

3. S1 Nuclease Digestion:

mRNA

123

Full Length Probe

Digested Probe

S1 Digestion

Add 300µl of: 0.3M NaCl, 5mM ZnSO4, 50mM sodium acetate, pH 4.5; containing 300-500 Units of S1 nuclease and 6µg calf thymus DNA (denatured). Incubate at 30°C for 30-60 minutes. Stop the reaction with 100µl of: 4M ammonium acetate, 25mM EDTA, 40µg/ml tRNA. Add 1 ml ethanol, precipitate the DNA and redissolve in 5µl of TE. Analyze 4µl on a denaturing PAGE gel (Section 2.1.2).

Figure 2.1.9a S1 mapping of a transcription start site. The length of the labeled probe fragment remaining after digestion reflects the distance between the 5’ end of the probe and the 5’ end of the RNA (the start site). Lanes: 1) Experimental, 2) Probe control, 3) Sequence ladder.

S1 mapping can also find intron sites (Figure 2.1.9b). In this case, the probe is derived from genomic DNA, and again labeled so that the labeled 3’ end falls within a coding portion of the gene. Any intron in this construct will not find a homologous region in the RNA, and will be cleaved by the S1 nuclease. In this case, the size of the labeled remainder reflects the distance from the label site to the splice site. Labeled Probe

mRNA

123

Full Length Probe

Products for S1 Mapping UreaGel-6 EC-836 Convenient system for casting 19:1 6% denaturing gels. Contains acrylamide, bis, TEMED, TBE and urea. (pg. 12)

TBE 10X EC-860 Formulated with 18 MegOhm water. 0.2 micron filtration. Costs less than bench-top buffers. (pg. 20)

Ammonium Persulfate EC-504 Exceeds ACS Standards. Low absorbed water results in consistent initiating capabilities. (pg. 32)

Tris - ULTRA PURE EC-406 Purified to remove ammonia and amine contaminants. Specifications include >99.9% purity. (pg. 33)

TE Buffer (100X) EC-862 100X Concentrated solution of 1M Tris-HCl, pH 8, with 100mM EDTA. 0.2 micron filtration.

EDTA - ULTRA PURE EC-610 Molecular biology grade EDTA. Specifications include low insolubles (<0.005%) and >99% purity. (pg. 32)

(pg. 20) Digested Probe

S1 Digestion

Figure 2.1.9b S1 mapping of an intron site. The nuclease digests the unhybridized intronic DNA. The size of the labeled fragment remaining indicates the distance between the 5’ end of the probe and the intron position. Lanes as in figure 2.1.9a. USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Applications

Electrophoresis

Ribonuclease Protection

Another procedure, ribonuclease protection, uses uniformly labeled RNA probes to analyze sample RNA. In this case, probes are chosen to fall entirely within the coding region, so they are only digested if no homologous RNA is present. Because the probes are uniformly labeled, the sensitivity of this technique is much higher than that for S-1 mapping. An excess of probe is mixed with the sample to be analyzed, and the hybrids are digested with RNase A, which will digest only ssRNA. The amount of probe protected from digestion (because it has hybridized with target RNA) is quantified by denaturing gel electrophoresis followed by autoradiography or by running on an automated sequencer. One of the strengths of this technique is that multiple probes can be added to a single sample, provided that they are of different sizes. A single reaction can thus give quantitative information on many different RNA species. Probe+mRNA

mRNA plus probe

Probe-mRNA

Probe (control)

hybridization

Primer Extension

Primer extension is another technique used to analyze RNA structure and expression. In this method, an oligonucleotide primer is annealed to RNA and extended to a cDNA copy by reverse transcriptase in the presence of labeled dNTPs. Alternately, the primer is labeled and no label is included in the extension reaction. If the RNA of interest is present, extended products will appear on a denaturing gel. Furthermore, the size of the extended product will indicate the position of the 5’ end of the RNA, and, if an excess of primer is used, the amount of cDNA produced will reflect the amount of target RNA in the sample. Primer extension provides the same type of information as S1 mapping. However, primer extension is unaffected by splice sites. In cases where only a genomic probe is available and an intervening splice site prevents S1 mapping of the start site, primer extension offers a useful alternative. Primer extension offers additional advantages over S1 mapping. A genomic clone of the target RNA is not required, and only 30 - 50 bases of sequence need be known to generate the primer. Additionally, probe preparation is easier because the primer is single stranded.

digestion Extended Primer mRNA plus primer

Figure 2.1.9c In RNase Protection, an excess of labeled probe is hybridized to the mRNA pool. Digestion with RNase followed by gel electrophoresis (Probe + mRNA) provides quantitation of the amount of probe complementary mRNA expressed. After digestion in the absence of mRNA (Probe - mRNA) no probe remains.

Primer

Protocol 2.1.9b

Figure 2.1.9d I n P r i m e r Extension, the probe introduced to the mRNA pool will hybridize with the RNA of interest if it is present. Hybrids are then extended by reverse transcriptase.

Ribonuclease Protection NOTE: ALL GLASSWARE AND REAGENTS MUST BE RNase FREE OR DEPC TREATED. 1. Probe Preparation:

The RNA probe is prepared by in-vitro transcription of a cloned DNA fragment. The DNA must be cloned into a vector which provides a promoter for T-7 RNA polymerase. To 0.5 µg of substrate in 1µl, add: 4µl of buffer, 1µl 200mM DTT, 2µl of NTP mix, 10µl 32P-CTP, 1µl (30U) placental RNase inhibitor.

Buffer: 200mM Tris HCl, pH 8, 40mM MgCl2, 10mM spermidine, 250mM NaCl.

NTP mix: 4mM each of ATP,GTP, and UTP in 0.5mM EDTA.

Mix the solution well and add 10 units of RNA polymerase (T7 or T3) in 1µl. Incubate at 37°C for 30-60 minutes.

Add 10 Units of (RNase free) DNase I. Incubate at 37°C for 15 minutes.

Add 30µl of 1µg/µl tRNA, phenol extract and ethanol precipitate 3 times to remove unincorporated label.

Redissolve the pellet in 100µl of hybridization solution: 80% formamide, 40mM PIPES pH 6.4, 400mM NaCl, 5mM EDTA.

Count 1µl to confirm 109 cpm/µl.

2. Hybridize probe and sample:

Dissolve 10µg dried sample RNA in 30µl of probe solution. Mix by pipetting until all RNA is dissolved. Denature at 84°C for 5 minutes. Transfer to a bath at 35-60°C and incubate for 6-24 hours. Time and temperature must be optimized for each probe.

3. Digest unbound probe:

Add 350µl of 20mM TrisHCl pH 7.5, 300mM NaCl, 5mM EDTA, 35µg/ µl RNase A, 3µg/µl RNase T1. Incubate for 30 min. at 30°C. Add 10µl 20% SDS and 3µl 25mg/ml proteinase K to digest the RNase. Incubate at 37°C for 20 minutes.

Phenol extract and ethanol precipitate. Redissolve the pellet in 5µl and run on a denaturing PAGE gel. USA: 1-800-526-3867 EUROPE: 441 482 646022

Protocol 2.1.9c Primer Extension 1. Primer Selection and Preparation:

Select a priming site that is 30 - 50 bases long, containing no self complementary sequences. The site should be within 150 bases of the transcriptional start site, as reverse transcriptase has a tendency to find pause/termination sites in larger transcripts. End-label the primer using 32P ATP and T4 polynucleotide kinase. Use the buffer and protocol recommended by the enzyme supplier for best results. Labeling of 100 µg of primer should incorporate 1-5x107 cpm, or 3x105 cpm/µg. Remove unincorporated label by 3 rounds of precipitation with 1 volume 4M ammonium acetate and 10 volumes ethanol. Precipitate for 30 minutes @ -70°C, and redissolve in 30 µl water between precipitations.

2. Hybridization: To 50 µg RNA sample in 100 µl, add 0.1µg (3x104 cpm) of labeled probe. Add 0.1 volume 3M sodium acetate, and 2.5 volumes Ethanol, and precipitate for 30 minutes at room temperature. Pellet, remove supernatant and allow pellet to air dry for 15 minutes. Over drying will make redissolving the pellet difficult.

Redissolve RNA/probe in 30 µl of hybridization buffer (3M NaCl, 0.4M HEPES pH 7.6, 1mM EDTA).

Hybridize overnight at 30 - 50°C (optimize temp. to reduce background).

Precipitate 30µl hybridization with 150µl 0.3M sodium acetate and 500µl of Ethanol. Wash pellet with 70% ethanol containing 30mM sodium acetate pH 5.3. Remove supernatant and allow pellet to air dry 15 minutes.

3. Primer Extension Reaction: Redissolve sample pellet in 18 µl H2O, 2.6 µl 10X RT buffer, 3.5 µl 4mM dNTP’s, 2 µl RNase inhibitor. Add 400 units of reverse transcriptase (AMV). Allow reaction to proceed at 42°C 1.5 hours. Stop reaction with 1 µl 500 mM EDTA.

Digest substrate RNA with 1µg (1 µl of 1µg/µl) RNase A to prevent gel distortions. Digest 1 hour at 37°C. Extract reactions with phenol and then ethanol precipitate.

Redissolve in 5 µl of water, add denaturing loading buffer and analyze 2-5 µl on a denaturing PAGE gel.

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

2.1.10 Analysis of DNA/Protein Interactions

The binding of proteins to specific DNA sites is an important mechanism of cellular regulation. Numerous techniques have been developed to analyze the interactions of regulatory proteins with DNA. Three techniques are presented below which analyze the site of protein binding on the DNA (“Footprint” Analysis).

DNase I Footprinting 1. Preparation of DNA substrate: DNA to be analyzed must be cloned in such a way as to present a restriction site for an enzyme leaving a 5’ overhang (3’ recessed OH) 50-150 bp from the putative binding site. This site is labeled by “filling in” the recessed site with 32P-dNTP’s using DNA polymerase. The probe is then cut from the remaining plasmid by a second restriction enzyme, 150 bases on the opposite side of the binding site. This releases a 200-300 bp probe, labeled at one end (see figure below).

Applications

Electrophoresis

A DNA binding protein will attach itself only to a specific, characteristic sequence on the DNA, and once bound, such a protein occludes the access by other proteins to the binding site. This regional protection is known as a protein’s “footprint.” The footprint of a particular binding protein may be accurately determined by treating a DNA-protein complex with the enzyme DNase I at low concentrations. DNase I will introduce “nicks” (single strand cuts) in the double stranded DNA, everywhere but in the footprint region. If the DNA is end labeled with 32P, the labeled fragments produced will end at a range of sites which covers the entire starting DNA molecule, excluding the footprint area (Figure 2.1.10a). Separation of these fragments on a denaturing gel yields a ladder of fragments with a gap corresponding to the footprint site. A Maxam & Gilbert sequencing ladder run as a control will allow assignment of the footprint with 1-2 base accuracy.

Protocol 2.1.10a

Protein Binding Site Restriction site

In addition to mapping the site of the footprint, it is possible to determine which residues within the site are important for binding. This is done using uracil or methylation interference. In these techniques, the nucleotides in the binding site are modified at an average of one site per molecule. This is done either with a methylation reaction which modifies guanine and adenine residues, or by synthesis in the presence of dUTP which substitutes uracil for thymine. The modified fragments are bound to the protein and run on a mobility shift assay (Section 2.2.4) which separates bound DNA from unbound DNA. Bound and unbound fractions are separately treated with reagents which cleave the DNA at the modified bases. As with DNase I mapping, end labeled DNA is used, and the resulting fragment lengths reflect the positions of modified bases. Bases which are not found to be modified in the bound fraction are important for protein binding.

Figure 2.1.10b Preparing the DNA substrate for DNase footprinting analysis. A circular construct containing the protein binding site is linearized with a restriction endonuclease, yielding two free ends, which are both labeled. One end is then cut away in a second round of restriction digestion, leaving an end labeled probe which carries the binding site.

Cleavage

Label ends using Klenow fragment

Restriction site

DNase I footprinting

Cleavage Maxam & Gilbert Sequencing Ladder

DNase I

Footprint Region

Binding Protein

Digest 5-10 picomoles of plasmid (10-20µg of a 3000bp construct) with an enzyme which will leave a recessed 3’ end 50-150bp from the binding site.

Ethanol precipitate the DNA, and wash 1X with 70% Ethanol.

Add 50 µl of 1X Klenow buffer containing 50 µCi of each 32P dNTP. Add 10 units of Klenow fragment in < 2 µl, and incubate 30 minutes at 25°C.

Klenow buffer : 50mM Tris HCl, pH 7.6 12mM MgCl2 1mM DTT 50 µg/ml BSA Chase reaction with 5 µl of 2mM each dCTP, dGTP, dTTP, dATP, to ensure complete polymerization.

Footprint Region

Label

Figure 2.1.10a A method for determining the location of a protein binding site, DNase I Footprinting Analysis involves endonuclease treatment of an end labeled DNA fragment bound to a protein. Limited digestion yields fragments terminating everywhere except in the footprint region, which is protected from digestion.

Ethanol precipitate twice with 0.1 vol 3M sodium acetate and 3 volumes of ethanol, or purify with a spin column or glass powder elution to remove unincorporated label.

Cut with a restriction enzyme to release a 200-300 bp end labeled probe.

Run probe on a 1.5-2% Agarose gel (Section 2.4.1) and recover fragment by electroelution (Section 2.4.3). Further purification may be necessary to ensure consistent results. Ion exchange mini columns provide good results.

If volume is >50µl, ethanol precipitate the probe, and reconstitute in 50 µl TE buffer.

Related Products AquaPor LE EC-202 General purpose agarose, AquaPor LE, combines excellent performance, gel strength and low background. (pg. 16)

UreaGel-6 EC-836 Convenient system for casting 19:1 6% denaturing gels with Acrylamide, Bis, TEMED, TBE and urea. (pg. 12)

continued

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Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Applications

Electrophoresis

2. Bind Protein to DNA Probe: Mix 105 cpm of probe with 200 µl of DNase Footprinting Buffer: 10mM Tris HCl, pH 7.5 4mM MgCl2 1mM CaCl2 150mM KCl 2mM DTT 100 µg/ml BSA 2mg/ml calf thymus DNA pH 7.5 (adjust buffer if necessary)

Add 20 µl protein sample. A series of dilutions covering 4-5 orders of magnitude will allow calculation of the binding affinity.

Prepare a blank tube with 20 µl of assay buffer

Incubate 30-45 minutes, at equilibration temperature.

The optimum time and temperature must be determined for each DNA/ probe combination.

Interpretation: Figure 2.1.10c shows an electrophoresis gel of idealized results. Bands correspond to DNase I cleavage sites. As the amount of protein present increases, the footprint area is progressively protected from cleavage. The concentration of protein required to give 50% protection can be mathematically related to the equilibrium constant for protein binding. See CPMB Section 12.4 for a complete discussion of this method of analysis.

3. DNase I Treatment:

DNase treatment proceeds for only 2 minutes, so stop solution and a dry ice Ethanol bath must be prepared before beginning the treatment.

Stop Solution: 6.5 ml ethanol 50 µl 1mg/ml tRNA 0.5 ml ammonium acetate saturated solution Cool to -70°C prior to use. Prepare DNase I solution:

The amount of DNase I required will vary depending upon the purity, age and storage conditions used for the enzyme. Start with 0.1 mg/ml DNase I and adjust to get an average of 1 nick per DNA molecule.

Dissolve DNase I in assay/equilibration buffer without BSA or calf thymus DNA. To each 200 µl sample of protein/DNA, add 5 µl DNase I solution.

Reproducible pipetting is essential at this stage if different DNA/protein ratios are to be compared.

Incubate at equilibration temperature exactly 2 minutes, then add 800 µl Stop Solution @ -70°C.

Precipitate DNA at -70°C for 30 minutes. Wash pellet with 70% Ethanol twice, and remove all supernatant. Air dry or speedvac 10 minutes.

Redissolve pellet in 6 µl of loading buffer, and run on a standard denaturing PAGE gel.

Convenient Products for Denaturing Electrophoresis UreaGel-6 EC-836 Convenient system for casting 19:1 6% denaturing gels. Contains acrylamide, bis, TEMED, TBE and urea. (pg. 12)

TBE 10X EC-860 Formulated with 18 MegOhm water. 0.2 micron filtration. Costs less than bench-top buffers. (pg. 20)

Ammonium Persulfate EC-504 Exceeds ACS Standards. Low absorbed water results in consistent initiating capabilities. (pg. 32)

Glass Free EC-621 Coats glass plates to prevent binding of polyacrylamide gels, allowing easy disassembly of the cassette. (pg. 31)

Autofluor LS-315 Water soluble scintillation phosphor for use as an autoradiographic image intensifier. (pg. 30)

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Figure 2.1.10c An electrophoresis gel showing successive DNase footprinting reactions conducted with increasing titrations of binding protein.

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Methylation Interference Assay Uracil Interference Assay

Probe Preparation for Methylation Interference:

Generate a probe labeled at one end from a plasmid construct digested with enzymes to produce one 5’ overhang on a probe of 100-300 bp. (see Protocol 2.1.10a, DNase I footprinting)

Prepare and purify fragment as described.

Methylate 106 cpm of probe in 200 µl of reaction buffer: 50mM sodium cacodylate pH 7.9 1mM EDTA

Add 1µl of DMS to start the reaction.

React for 5 minutes at room temperature.

Stop the reaction with 50 µl of: 1.5M sodium acetate, pH 7.2 1mM BME 0.25 µg/ml tRNA

Precipitate the DNA with 750µl of ethanol, at -70°C (dispose of supernatant as DMS toxic waste).

Wash pellet by redissolving in 300µl 0.3M sodium acetate, then precipitate with 900 µl ethanol. Repeat this step twice.

Proceed to mobility shift analysis.

Probe Preparation for Uracil Interference:

Binding site Labeled substrate

Base Modification

Modified bases

Applications

The two techniques differ in the base(s) targeted, and in the method used to modify and cleave the DNA. The methylation interference assay is the simpler of the two, involving a chemical modification of Guanines and Adenines with Dimethylsulfate to produce N-7 methyl G or N-3 methyl A residues. These residues are subject to cleavage by piperidine. The complexity of this method is somewhat increased by the need to isolate an end labeled probe with which to work. In the Uracil interference analysis DNA is synthesized in the presence of dUTP to incorporate Uracil residues in place of thymine, at a rate of 0.5-1 thymine substitutions per molecule. This can be accomplished by PCR with one labeled primer, thus probe generation may be easier than for methylation interference. Cleavage at Uracil residues requires a two step procedure, in which Uracil glycosylase removes the Uracil base, creating apyrimidinic sites which are then cleaved by piperidine.

Methylation Interference Assay Uracil Interference Assay

Electrophoresis

These two analytical methods are presented in parallel, because they assay for similar information based on similar reactions. In both cases, the DNA is analyzed for nucleotides which are important for protein binding. The approach taken is to end label the DNA probe, so that cleavage of the DNA will yield labeled fragments whose size indicates the cleavage position, as in DNase I footprint analysis. The probe is treated to generate modified bases at about 1 base per DNA molecule. Binding protein is added to the modified DNA. If the base which was modified on a given DNA molecule was critical for binding, that molecule will be left unbound. Bound and unbound populations of DNA are separated on mobility shift assay gels (Section 2.2.4). The two populations are then treated to cleave the DNA at the modified bases, and run on a denaturing PAGE gel. Modifications to bases important for protein binding will lead to the absence of cleavage fragments ending at such bases from the bound fraction of DNA. Cleavage at these sites will produce fragments seen only in the unbound DNA (Figure 2.1.10d).

Protocol 2.1.10c

Probe for this procedure is generated by PCR amplification in the presence of one labeled and one unlabeled primer, with dUTP present at 25% of the concentration of the dTTP. Primers should be selected to provide a region of amplification which is 200-300 bp in length, and contains the protein binding site. Label the primer(s) with 32P ATP and polynucleotide kinase. Use the buffer and protocol recommended by the enzyme manufacturer. Purify the probe by gel electrophoresis or spin columns prior to use. For PCR guidelines, see Section 2.2.3. Purify the PCR products by native gel electrophoresis (Section 2.2.5). Proceed to mobility shift analysis.

Mobility Shift Analysis:

This step is the same for both protocols, and is described in Section 2.2.4. At the end of the analysis, locate the bands by autoradiography, and cut out the bound (upper) and unbound (lower) DNA bands. Purify the DNA from the gel slices (section 2.2.5).

Cleavage Reactions: Protein binding

Not essential for binding

Uracil glycosylase (Uracil interference only)

Mobility shift assay

Remove the Uracil bases by treating the DNA with 0.02 U/µl Uracil glycosylase in 1X Taq polymerase buffer (from PCR reaction, Section 2.2.3). Incubate at 37°C 1 hour.

Precipitate DNA with 0.1 vol 0.3M sodium acetate and 3 volumes of ethanol.

Proceed with piperidine cleavage.

Piperidine Reaction: (both assays)

Fragments representing cleavage at a base essential for protein binding are only present in the unbound sample.

Bound Population

Unbound Population

Modified base prevents binding (not found in bound population)

Redissolve precipitated DNA in 0.1 ml of 1M piperidine.

Unbound due to binding equilibrium (also present in bound population)

Heat to 95°C for 30 minutes. Remove tubes to -70°C.

Lyophilize frozen samples to dryness.

Redissolve in 0.1 ml water.

Repeat lyophilization and rehydration three times.

Cleavage at modified bases

Figure 2.1.10d In uracil or methylation interference assay, bases critical for protein binding will not appear as bands in the bound population.

Analyze bound and unbound samples on a 6% denaturing polyacrylamide gel.

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Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Applications

Electrophoresis

2.2 Native Polyacrylamide Gel Electrophoresis of DNA In the absence of denaturants, double stranded DNA retains its double helical structure, which gives it a rodlike form as it migrates through a gel (for non-denaturing electrophoresis of single stranded DNA, see section 2.3.2, SSCP Analysis). Double stranded DNA of up to 1000 bp can be separated on polyacrylamide gels. DNA over 1kb is generally fractionated on agarose gels (Section 2.4). As with single stranded DNA, double stranded DNA has a uniform negative charge density imparted by the phosphate linkages in its nucleotide backbone. As a result it has a free solution mobility (in the absence of any sieving matrix) which is independent of the size of the molecule. In a sieving medium, though, such as polyacrylamide or agarose, the relative mobility of any given molecule is determined by its ability to find a path through the gel pores. This is a linear function of the effective radius of the molecule, which can be empirically demonstrated to be related to the log of the molecular weight. Thus, on a gel of particular porosity, the migration rate of a given fragment is inversely proportional to the log of its molecular weight (log mw).

Figure 2.2a Electrophoretic mobility versus log mw.

Finally, tracking dyes are added, typically xylene cyanole and bromophenol blue. These dyes migrate through the gel unsieved. The table below presents the DNA fragment sizes which co-migrate with these dyes in various percentage 29:1 PAGE gels. Effective Separation Ranges (bp) and Tracking Dye Co-Migration vs. Gel Percentage (Native 29:1 Acrylamide:BisAcrylamide) Gel %

Gel %

Bromophenol Blue (nucleotides)

Size Range (bp)

4 6 8 10 12

1000-2000 70-450 60-400 50-300 40-200

Xylene Cyanole (nucleotides)

95 60 45 35 20

450 240 160 120 70

Table 2.2.1a

2.2.2 Gel Preparation

Native PAGE gels are prepared by mixing an Acrylamide/Bisacrylamide monomer concentrate (AccuGel 19:1 or 29:1), buffer concentrate and water to achieve the desired gel concentration. TEMED and Ammonium Persulfate are added to initiate polymerization and the solution is poured into a cassette. The comb is then inserted. The cassette is formed by two glass plates separated by spacers, typically 0.5-1mm in thickness, and sealed at the bottom (Figure 2.2.2a). A more detailed discussion of the electrophoresis apparatus can be found in Section 1.5. The polymerization process takes 1-2 hours to complete. Figure 2.2.2a An exploded view of a gel cassette.

Casting Solution

Comb Spacer

The proportionality of log mw with mobility holds within certain limits. The very smallest molecules are not sieved effectively by the gel and all migrate at the same rate. Similarly, the largest DNA molecules cannot determine a path through the gel, and all molecules above this size migrate at the same near zero rate. The mobility of double stranded DNA may also be affected by the sequence of bases. Certain sequences lead to bending of the helical structure, which can cause anomalously slow migration of the fragment. (Such altered mobilities serve as a basis for separation in Heteroduplex Analysis (Section 2.3.1)). These anomalies cause problems for the determination of fragment molecular weight by native PAGE, which can be much more reliably assigned with denaturing PAGE. DNA PAGE gels are generally run in TBE Buffer, which has a high buffering capacity. TAE may also be used, but its lower buffering capacity leads to buffer exhaustion and pH changes in the gel during protracted runs. The voltages used are relatively low, around 5V/cm. With higher voltage, larger DNA molecules show a disproportionate increase in mobility, leading to a compression of bands in the higher size range.

2.2.1 Sample Preparation

Sample preparation for native PAGE is straightforward. Since the DNA does not need to be denatured, the concern is mainly with buffer content, density and visibility. The salt concentration of the sample should be no greater than the 200mM salt concentration in the running buffer, because gross imbalances in salt content between sample and gel can lead to salt waves, band distortions and smearing. The density of the sample mixture is adjusted to ensure that the samples remain in the wells prior to electrophoresis. Generally sucrose or ficoll is used for this purpose, as these uncharged compounds will not run into the gel or interfere during electrophoresis. Glycerol should not be used, because it forms complexes with the Borate in TBE buffer. These complexes can migrate into the gel and distort the band pattern. 56

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Plates

1. Prepare solution of monomer, buffer and water as per table below. 2. De-gas if desired. 3. Add 0.8ml of 10% APS and 0.1ml TEMED per 100ml solution. 4. Pour gel. Formulas for Native DNA Polyacrylamide Casting Solutions using National Diagnosticsâ&#x20AC;&#x2122; AccuGel 29:1 (40%) Gel %

TBE Gels

TAE Gels

TTE Gels

AccuGel: 15ml 10X TBE: 10ml di H2O: 75ml

AccuGel: 15ml 50X TAE: 2ml di H2O: 83ml

AccuGel: 15ml 20X TTE: 5ml di H2O: 80ml

AccuGel: 20ml 10X TBE: 10ml di H2O: 70ml

AccuGel: 20ml 50X TAE: 2ml di H2O: 78ml

AccuGel: 20ml 20X TTE: 5ml di H2O: 75ml

10%

AccuGel: 25ml 10X TBE: 10ml di H2O: 65ml

AccuGel: 25ml 50X TAE: 2ml di H2O: 73ml

AccuGel: 25ml 20X TTE: 5ml di H2O: 70ml

12%

AccuGel: 30ml 10X TBE: 10ml di H2O: 60ml

AccuGel: 30ml 50X TAE: 2ml di H2O: 68ml

AccuGel: 30ml 20X TTE: 5ml di H2O: 65ml

Table 2.2.2a Add 0.8 ml of 10% APS and 0.1 ml of TEMED per 100 ml solution.

Gels are run in 1X buffer, at 5V/cm for 1-2 hours, until the Bromophenol Blue tracking dye has reached the end of the gel, or until it is known that the fragments of interest have migrated about 50% through the gel. Monitor upper and lower tank pH during the run if TAE is used and replenish buffer if pH begins to drift.

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Applications of Native DNA PAGE 2.2.3 PCR Analysis

Extension o 72 C Primer Annealing o 50 C

Figure 2.2.3a One cycle of the polymerase chain reaction (PCR). The double stranded substrate is denatured at 95°C. Primers are annealed to the single strands at 50°C, and extended at 72°C to produce two copies of the original template.

An important aspect of PCR is its ability to amplify specific sequences from complex mixtures. Given primers of sufficient specificity, and with some effort at optimizing conditions, a single sequence can be isolated from total RNA or genomic DNA using this technique. This allows rapid cloning of target sequences without creating and screening a library. The flanking sequences needed to design the primers can often be derived from end sequencing of proteins, from knowledge of gene structure (i.e. using the poly A of RNA as a priming site) or from conserved regions in homologous sequences. Ideally, a PCR reaction gives one product of a predicted size, which needs only to be separated from the unused primers and dNTPs. In practice, particularly in amplifications from complex mixtures, multiple products are often produce d. One of the most common artifacts is “primer-dimer”. This is produced when the primers used are able to hybridize to each other at their 3’ ends. These hybrids are extended efficiently into a 30-50 bp structure, which competes for amplification with the target DNA. The result is a low molecular weight band, which in the worst cases is over 90% of the reaction product. Products for Native DNA PAGE AccuGel 29:1 (40%) EC-852 Stabilized, pre-filtered 40% acrylamide: bisacrylamide solution. Two year shelf life-at room temperature. (pg. 15)

AccuGel 29:1 (30%) EC-851 Stabilized, pre-filtered 40% acrylamide: bisacrylamide solution. Two year shelf life-at room temperature. (pg. 15)

TBE 10X EC-860 Formulated with 18 MegOhm water. 0.2 micron microfiltration. More economical than bench-top buffers. (pg. 20)

TAE 50X EC-872 Ultra-Pure preformulated buffer for electrophoresis. TAE optimizes sample recovery. (pg. 20)

Ammonium Persulfate EC-504 Exceeds ACS Standards. Low absorbed water results in consistent initiating capabilities. (pg. 32)

TEMED EC-503 Fractionally distilled twice to remove absolutely all inhibitory and fluorescent contaminants. (pg. 33)

Applications

Figure 2.2.3b Mis-priming can produce unintended products. After a single round, any mismatches between the primer and the improper sequence will be eliminated, as the figure at left shows. The improper target will have no impedance to its amplification along with the target DNA.

Annealing of the primer to a non-target site attaches non-target DNA (gold) to authentic primer sequence (blue)

Denaturation o 95 C

Electrophoresis

The polymerase chain reaction (PCR) is a powerful technique which uses repetitive cycles of primer annealing, primer extension, and product denaturing to produce an exponential increase in the copy number of the target DNA. Two primers are used, which flank the region of interest (Figure 2.2.3a). In the presence of a thermostable polymerase, the substrate DNA is denatured at 95°C, the primers are annealed at 50°C, and the polymerase generates new copies of the DNA at 72°C. In the next cycle, twice as many molecules are available for primers to anneal to; thus for n cycles, 2n products are produced. Typically 30 cycles are used, which generates 230 = 1 X 109 copies from one original. For a 500 bp fragment, this corresponds to about 1 pg of DNA from each copy, or a microgram of DNA from each femtogram of starting material.

Nontarget bands are also produced from mis-priming. If the primers have sufficient homology to some nontarget DNA region, this region will be amplified. It is important to realize that mismatches between primer and target only impede the first round of amplification. Once the first product has been copied, a perfect match is generated for the next annealing (Figure 2.2.3b). Mis-priming is minimized by using the highest annealing temperature which gives product, and by optimizing the Mg+2 concentration in the reaction. It is also important to avoid over-amplification. Once a product has accumulated to >1µg/reaction, the probability of one of the primers finding an unintended priming site within the target sequence is greatly increased. This generates a truncated fragment which is more efficiently amplified than the longer target. Because this smaller fragment contains a portion of the target sequence, it can interfere with subsequent analysis of the reaction, such as sequencing or blot analysis.

Annealing of a second primer downstream of the first site initiates complementary strand synthesis.

Extension of the second primer produces a construct which contains non-target DNA between two authentic primer sequences.

In all subsequent cycles, the new construct will amplify at least as efficiently as the target sequence.

Protocol 2.2.3a PCR Amplification Protocols and conditions for PCR depend strongly on the enzyme, primers and substrate used. General guidelines for use of Taq polymerase are given below: Reaction mixture (25-100µl): 25-50µl of Taq Buffer 0.2 mM dNTP’s 0.5 µM each primer 2-3 units Taq Polymerase Target sequence Taq buffer contains 67mM Tris-HCl, pH 8.8, 150mM (NH4)2SO4, 1-5mM MgCl2, and 10mM BME. The concentration of Mg++ will vary from 1-5mM, depending upon primers and substrate. The amount of substrate used depends upon the concentration of target sequence in the sample DNA. The target will be amplified by up to 106 fold in a successful reaction, but the amplification will usually plateau at 1-10µg. Thus, 1 pg of target sequence in the reaction is a good place to begin. Prepare the reaction in an 0.5ml microcentrifuge tube, and overlay it with 50-100 µl (1-2 drops) of mineral oil to prevent evaporation. Place the tube(s) in a thermocycler and run 20-30 amplification cycles. continued

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Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Gel Electrophoresis of PCR Products

Applications

Electrophoresis

Cycle Conditions:

Denature the reaction at 90-94°C for 0.5 - 1 minute. The time and temperature should both be the minimum compatible with product production. Taq polymerase has a half-life of 20 minutes at 94°C. Consequently, after 30 one minute cycles with denaturation at 94°C, more than half the enzyme activity will be lost.

After denaturing, anneal the primers at 45-60°C. This temperature is one of the most critical optimization parameters. Start at 5-10° below the lowest calculated melting temperature (Tm) of the primer pair, and increase for subsequent reactions until yield begins to decline. The annealing step requires 0.5 - 1 minute.

Finally, increase the temperature to 72°C, the optimal temperature for Taq polymerase. Allow the Taq to extend the annealed primers for 1.5 - 3 minutes. Many programs increase the extension time for each successive cycle, to compensate for lost Taq activity and increased substrate concentrations.

Cycle summary:

1. 2. 3. 4.

90 - 94°C 45 - 60°C 72°C Return to (1)

0.5 - 1 minute 0.5 - 1 minute 1.5 - 3 minute

30 such cycles are usually sufficient to amplify 1-10 µg of product. 35-45 cycles may be used, but internal priming on the product and over amplification of unwanted bands often result from over-cycling. Generally, it is better to focus on optimizing reaction conditions than to go beyond 35 cycles.

Gel electrophoresis of PCR products is the standard method for analyzing reaction quality and yield. PCR products can range up to 10kb in length, but the majority of amplifications are at 1kb and below, where PAGE analysis is the most effective. In the size range from 400 to 1000 bases, the choice of native PAGE or agarose for the analysis of PCR products depends mainly upon whether the product will need to be further purified. Purification from agarose is generally more convenient. Electrophoresis reveals the size of the product band, which is compared with the predicted result. Electrophoresis also shows how much of this band was produced, and reveals the presence or absence of any unintended amplification products. Section 2.4.1 discusses how to prepare an agarose electrophoresis gel and Section 2.4.3 discusses methods for purification from agarose. PAGE gels for PCR products are poured and run as described in Section 2.2.2. The concentration of acrylamide and the amount of cross-linker are chosen to give pore sizes optimal for the size of DNA fragment desired. See Table 2.2.2a for suggested gel percentages for a variety of fragment sizes. Gels are most often stained in Ethidium Bromide, even though the fluorescence of this stain is quenched by polyacrylamide, which decreases sensitivity 2-5 fold. This decrease in sensitivity generally does not present a problem, because most PCR reactions yield product levels in the microgram range, and Ethidium will detect as little as 1/10 of this amount.

Interpretation

Ideally, electrophoresis yields a single strong band of correct size, as determined by comparison with size markers run on the same gel. If possible, identity should be confirmed by digestion with a restriction enzyme with a known site in the target DNA, or by Southern analysis (Section 4.1.2). An unexpected band running below 100bp is usually primer-dimer. If this is the case, this band will also be found in the control reaction, run without substrate. Adjustment of Mg2+ concentration may help to minimize primer-dimer, but primer redesign may be required to eliminate it completely. Multiple bands from a PCR reaction are a bad sign. They indicate multiple priming sites for the primers within the target DNA, and call into question the reliability of the primer set. Often, multiple bands may be eliminated by raising the hybridization temperature. Sometimes adjusting the Mg2+ concentration eliminates unwanted products. Reducing the primer concentration will also make the annealing more specific, eliminating incorrect amplifications. As a final resort, band(s) which appear to be correct molecular weight can be cut out and purified (Section 2.2.5 for PAGE, Section 2.4.3 for agarose).

Products for PCR Analysis

Figure 2.2.3c PCR amplification follows an exponential curve until a saturation point is reached, after which further amplification often serves only to degrade purity. Curve shape depends upon the amount of substrate present initially.

Yield and Kinetics

PCR reactions produce product in a nonlinear pattern (Figure 2.2.3c). Amplification follows a typical exponential curve until some saturation point is reached. Generally products will not be further amplified once 1-5 µg has been generated. Saturation by one product of a reaction does not always prevent further amplification of other (generally unwanted) products. As noted above, this means that over-cycling may decrease the quality of an otherwise good reaction. When first optimizing a reaction, it is advisable to take samples every 5 or 10 cycles to determine the number of cycles actually needed. 58

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AccuGel 29:1 EC-852 Stabilized, pre-filtered acrylamide: bisacrylamide solution. Two year shelf life-at room temperature. (pg. 15)

Nuclistain EC-730 For the UV free visualization of DNA on acrylamide and agarose. Improves the recovery of PCR products. (pg. 29)

TBE 10X EC-860 Formulated with 18 MegOhm water. 0.2 micron filtration. More economical than bench-top buffers. (pg. 20)

TAE 50X EC-872 Ultra-Pure preformulated buffer for electrophoresis. TAE optimizes sample recovery. (pg. 20)

Ammonium Persulfate EC-504 Exceeds ACS Standards. Low absorbed water results in consistent initiating capabilities. (pg. 32) AquaPor LE EC-202 AquaPor LE is a high quality, general purpose agarose ideal for most routine applications. (pg. 16)

TEMED EC-503 Fractionally distilled twice to remove absolutely all inhibitory and fluorescent contaminants. (pg. 33)

AquaPor LM EC-204 AquaPor LM has the highest gel strength available for a low melt agarose.(pg. 16)

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

2.2.4 Mobility Shift Assay

The mobility shift assay gel uses a low ionic strength buffer system, to avoid salt effects on binding constants. This necessitates buffer circulation between upper and lower chambers to prevent buffer exhaustion and pH shifts during the run. 10X Gel Buffer: 67mM Tris HCl pH 8.0 33mM sodium acetate 10mM EDTA

Mix components and adjust pH to 8.0 Use this buffer at 1X for upper and lower buffer chambers.

Gel Mix: 6.7ml 30% acrylamide (AcrylaGel) 1.25ml 2% methylene bis-acrylamide (Bis-AcrylaGel) 1.25ml glycerol 36ml H2O 5 ml 10X gel buffer

Just prior to pouring, add 125µl 10% APS and 45µl TEMED to initiate polymerization.

Gel assembly:

Cast the gel in a standard format (16cm plate) cassette, with 1.5mm spacers and wells at least 0.8cm wide. Pour using standard techniques, and allow to polymerize one hour.

Run Conditions:

Recirculate the buffer at least 10ml/min.

Probe Preparation:

Pre-run gel for 1.5 hours at 6V/cm

A probe of the proper size is cut from 10 µg of plasmid clone, using restriction enzymes which will yield probe of 50-150 bp, with one 5' overhanging end.

Preload at least one well with 0.01% bromophenol blue in 10% glycerol, to provide a tracking dye. No dyes are included in the binding reactions.

Label the probe with 32P dNTP and Klenow fragment, to fill in the overhang. To the restriction digest reaction, add 100µCi of 32P dNTP (chosen to be part of the filled in region) and 0.2 mM of the other necessary dNTP’s.

Add 3 units of Klenow fragment and incubate 30 minutes @ room temperature.

Load binding reactions and run gel at approximately 12 V/cm for 1-2 hours. Adjust the run voltage so that plates do not become warm, because increases in temperature will alter binding equilibria. A current of 25-30 mA is sufficient for a 16 cm gel. Gels may be run at higher voltage in a cold room.

Add an additional aliquot of a mixture of all 4 dNTP’s to a final concentration of 0.2mM, and incubate another 10 minutes.

Add 0.1 volume 3M sodium acetate, 3 volumes ethanol, precipitate the DNA and wash once with 70% ethanol to remove the bulk of the unincorporated label.

Isolate the probe on an agarose gel by electrophoresis onto a DEAE membrane (Section 2.4.3)

Probe should be labeled to 107 - 108 cpm/µg.

Protocol 2.2.4a Mobility Shift Assay

Binding Reaction:

Results:

Bound DNA will appear as a more slowly migrating band, which is not visible in the lanes without protein. The band will disappear on addition of unlabeled competitive DNA sequences.

Products for Mobility Shift Assay AcrylaGel EC-810 Stabilized, pre-filtered 30% acrylamide solution. Two year shelf life at room temperature.

Bis-AcrylaGel EC-820 Stabilized, pre-filtered solution of 2% methylene bisacrylamide. Aldehyde and acrylic acid free. (pg. 15)

Ammonium Persulfate EC-504 Exceeds ACS Standards. Low absorbed water results in consistent initiating capabilities. (pg. 32)

TEMED EC-503 Fractionally distilled twice to remove absolutely all inhibitory and fluorescent contaminants.

(pg. 15)

Mix:

104 cpm of DNA probe 2µg nonspecific DNA (calf thymus DNA, or synthetic polymer) 20 µg sample protein 1 µl glycerol in a final volume of 15 µl

Incubate at 30°C for 15-30 minutes.

This gives a basic framework for a binding reaction. Components which may be needed to ensure binding include glycerol (10%), KCl (50mM) and DTT (1mM). Buffer conditions must be optimized for each protein/DNA combination studied. continued

Applications

In the mobility-shift (or gel-shift) assay, end labeled DNA is allowed to bind protein. The resulting DNA protein complexes are then run on a non-denaturing PAGE gel and the gel is dried and autoradiographed. The buffer composition of the PAGE gel is varied from the standard TBE gel, because lower ionic strength is needed to facilitate the DNA protein binding. It is interesting to note that binding interactions which can dissociate in free solution within 1 minute show altered mobility on gels which require more than an hour to run. It is theorized that the gel matrix forms a “cage” around the DNA-protein complex, which prevents the components from diffusing away from each other, thus promoting re-association and in effect stabilizing the complex. This would argue that the ratio of bound and unbound material seen on the gel is a direct measure of the fraction of bound material in the sample as it entered the gel. Thus, the conditions set in the binding reaction are of critical importance.

Electrophoresis:

Electrophoresis

Protein bound to a small piece of DNA will alter the electrophoretic mobility of that DNA fragment. This allows the analysis of protein-DNA interactions, including the measurement of binding rates, affinity, and specificity. In addition, bound and unbound DNA may be isolated from the gel and used for further types of analysis such as methylation interference or uracil interference (Section 2.1.10).

Glycerol - ULTRA PURE EC-606 Ultra Pure for molecular biology applications. Specifications include >99.7% purity. (pg. 33)

(pg. 33)

Tris - ULTRA PURE EC-406 Purified to remove ammonia and amine contaminants. Specifications include >99.9% purity. (pg. 33)

EDTA - ULTRA PURE EC-610 Molecular biology grade EDTA. Specifications include low insolubles (<0.005%) and >99% purity. (pg. 32)

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Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Applications

Electrophoresis

2.3 Conformational Analysis

Protocol 2.3.1a

Native DNA PAGE gels can be used to detect small mutational differences between DNA molecules. In heteroduplex analysis, for double stranded DNA, the basis of separation is the conformational difference arising from the bending of the rodlike double helix caused by small mismatches between the strands. In SSCP analysis, single stranded DNA molecules are fractionated based on the compactness of their folded structure. In both cases, it is possible to separate strands of equal length which have different sequences, often differing by only 1 base. Given a control (unmutated) fragment to use as a gel standard, these techniques can be used to screen large numbers of samples for mutations.

Heteroduplex Analysis GEL PREPARATION 1. Preparation of working solutions:

To cast a 0.8 to 1.0 mm thick gel (>40 cm vertical gel recommended) combine the following in an Erlenmeyer flask: 50 ml SequaGel MD 6 ml 10X TBE 15 g urea (optional)

2.3.1 Heteroduplex Analysis

Double stranded DNA is not a completely straight rigid rod. Sequence variations can cause bends in the double helix, or even alter the basic structure of the helix. A bend or kink in the DNA restricts its mobility through a sieving matrix because the bent molecule presents a larger projected area to the gel pores. A mismatch between the two strands of DNA in a duplex can produce a more radical kink in the structure, producing a heteroduplex species which can easily be resolved from the homoduplex by electrophoresis. In this system, control DNA is denatured and allowed to anneal with denatured sample DNA. The renatured products are analyzed on a gel optimized to resolve conformational differences, such as National Diagnostics’ SequaGel MD. If the sample DNA is not identical to the control DNA, multiple bands are observed. The fastest migrating band is the homoduplex control and/or the homoduplex sample. Heteroduplexes with mismatches migrate more slowly. Heteroduplex Analysis is easily applied to large numbers of samples, and is particularly suited to the analysis of PCR products, because both sample and control DNA must be of the same size. In PCR, this would correspond to using the same amplification primers.

Fill to 100 ml with deionized water and mix thoroughly. Urea may assist the formation of more distinct bands during electrophoresis and reduces the formation of doublets in homoduplex controls.

2. Casting the gel:

Treat one plate from the gel cassette with Glass Free to facilitate later disassembly of the cassette.

Add the following to the solution, and swirl gently: 40 µl TEMED 400µl freshly prepared 10% ammonium persulfate

Using standard acrylamide procedure (Section 2.1.2), pour the gel solution into the cassette, insert the comb, and allow to polymerize at room temperature for a minimum of 60 minutes. Attach the gel cassette to the electrophoresis apparatus, and fill the upper and lower chambers with 0.6X TBE.

SAMPLE PREPARATION 1. PCR amplification:

PCR conditions should be optimized for the desired PCR product before heteroduplex analysis. It is recommended that the minimum number of PCR cycles be used on a purified, salt free template, and that reagent and primer concentrations be optimized.

After PCR thermal cycling, add EDTA to a final concentration of 5 mM (1µl of 0.5 M EDTA per 100 µl reaction) to inactivate the Taq DNA polymerase.

2. Hybridization:

Wild type

Mutant

Denaturation

Mix equivalent quantities of wild type and sample PCR-amplified DNA. Heat at 95°C for 3 minutes. Then, over a 20-30 minute period, slowly cool the mixture to room temperature. The use of a thermocycler can facilitate this step.

ELECTROPHORESIS

Annealing

2. Rinse the wells with running buffer and load the samples in the 1.0X SequaGel MD gel. One lane should consist of control homoduplex DNA, and one of the sample homoduplex DNA. This will allow the detection of non-heteroduplex artifacts on the gel. Another lane should consist of an appropriate DNA size marker.

Duplexes

Wild Type

Mutant

1. Add 1 µl Triple Dye Loading Buffer (provided in kit) per 5 µl of sample and mix thoroughly.

Hetero-duplex Bands

3. Run the gel in 0.6X TBE, at a constant voltage of 20 V/cm, as determined by the length of the gel. For a 40 cm gel, set the power supply to 800 V. Approximate run times can be estimated from the chart below:

Homoduplexes

Heteroduplexes

Figure 2.3.1a Heteroduplex Analysis. Annealing of mutant DNA to wild type probe gives duplexes with one or more mismatched bases (heteroduplexes). Mismatching causes the double helix to take on a conformation which retards its mobility during electrophoresis.

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Fragment Size Run Time (800V) 200 bp 250 bp 300 bp 500 bp 700 bp 900 bp

14.0 hours 14.5 hours 16.5 hours 20.0 hours 25.0 hours 30.0 hours

Volt X Hours 11,200 11,600 13,200 16,000 20,000 24,000

4. When the electrophoretic run is complete, remove the gel from the apparatus, and carefully remove one plate from the gel. Stain with ethidium bromide or silver stain. continued

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Protocol 2.3.2a

STAINING

To visualize ethidium bromide stained bands, cover the gel with plastic wrap and place the plate with the gel side down on a UV-transilluminator. It may assist in handling and visualization to cut out the gel region containing the bands of interest.

WARNING: ETHIDIUM BROMIDE HAS BEEN SHOWN TO BE A CARCINOGEN AND SHOULD BE DISPOSED OF PROPERLY.

Silver staining (Section 4.1.1) may be used to increase band visibility.

2.3.2 SSCP Analysis

Single stranded DNA can adopt multiple conformations under non-denaturing conditions. In the absence of a complementary strand, DNA will anneal short internal complementary sequences, forming a complex “knot”. A DNA molecule follows a complex path of folding to reach its final form. This form is influenced by the solution environment and temperature. The path consists of a series of annealing steps, each stabilizing and, to some extent, directing the next step. Minor alterations in the sequence of the DNA will disrupt the annealing process and result in a different final shape. The compactness of these structures will determine how fast the single stranded DNA migrates through a non-denaturing gel. Such differences in electrophoretic mobility between nearly identical strands serve as the basis for the technique known as single strand conformation polymorphism (SSCP) analysis. Like heteroduplex analysis, SSCP is a rapid method for mutational screening.

Wild type Wild Type

Denaturation

Mutant

Mutant + Wild Type

Samples are denatured with heat and then rapidly cooled. Rapid cooling favors self-annealing, because insufficient time is allowed for complementary strands to collide and orient for duplex formation. The renatured samples are analyzed on a gel opposite the control DNA. As with Heteroduplex Analysis, all fragments must be the same length. Mutant samples will show a mobility different from the control DNA. The gel matrix used must be optimized for the resolution of DNA conformers of the same length. Various combinations of Acrylamide/ Bis-Acrylamide are mentioned in the literature, at ratios from 29 to 1 to 50 to 1, and at percentages from 4 to 8. National Diagnostics’ SequaGel MD (page 12) is optimized to provide superior results in both heteroduplex and SSCP analysis.

GEL PREPARATION 1. Preparation of working solutions:

To cast a 0.4 mm thick gel (>40 cm vertical gel recommended) combine the following in an Erlenmeyer flask: 25 ml SequaGel MD 6 ml 10X TBE Fill to 100 ml with deionized water and mix thoroughly.

Prepare 0.6 X running buffer by diluting 60 ml of 10X TBE stock to 1 L with deionized water.

Applications

SSCP Analysis

Electrophoresis

Stain using 0.6X TBE containing 1 µg /ml of ethidium bromide. Water should not be used in place of the TBE, because the gel will swell when placed in water. Stain for 15-30 minutes. For maximum sensitivity, destaining in 0.6X TBE for up to 30 minutes may be required.

2. Casting the gel: Add the following to the gel solution, and swirl gently: 40 µl TEMED 400 µl freshly prepared 10% APS

Using standard acrylamide procedures, pour the gel solution into the cassette, insert the comb (inverted if using a sharkstooth comb), and allow polymerization at room temperature for a minimum of 60 minutes. Attach the gel cassette to the electrophoresis apparatus.

SAMPLE PREPARATION 1. PCR amplification: PCR conditions should be optimized for desired PCR product before SSCP analysis is undertaken. It is recommended that the minimum number of PCR cycles be used on a purified, salt-free template, and that reagent and primer concentrations be optimized. If radiolabeling is going to be utilized instead of silver staining, end-labeled primers may be used, or an α-32P dNTP may be included in the PCR amplification. 2. After PCR thermal cycling, 1 µl of PCR product should be added per 10 µl of SCCP Stop Solution (provided with SequaGel MD kit). To denature the sample DNA, this solution should be heated to 94°C for 2 minutes. The vials should then be placed immediately into an ice slurry to rapidly cool the solution. ELECTROPHORESIS 1. Rinse the gel wells with running buffer. The sharkstooth comb should be reinserted so that it just touches the surface of the gel, and 1 to 3 µl of the sample should be loaded. 2. Run the gel at a constant power of 6-8 watts for 14 hours. 3. If the DNA was radiolabeled, transfer the gel to Whatman 3MM filter paper, place on a flat surface, and cover with plastic wrap. Dry the gel and, using standard technique, expose to X-ray film. Silver staining may be used for detection. Ethidium bromide is not effective at detecting single stranded DNA.

Products for Conformational Analysis

Annealing

Figure 2.3.2a SSCP Analysis (Single Strand Conformational Polymorphism). Single point mutations can cause major differences in the folded form of single stranded DNA. These differences can be detected as differences in electrophoretic mobility.

SequaGel MD Monomer Solution EC-845 For the detection of minor mutational differences in SSCP Analysis and Heteroduplex Analysis. (pg. 14)

TBE 10X EC-860 Ultra-Pure, Convenient, and Economical. Formulated with 18 MegOhm water. 0.2 micron microfiltration. Costs less than bench-top buffers. (pg. 20)

SequaGel MD Heteroduplex Kit EC-847 Contains SequaGel MD Monomer Solution (200ml) and Triple Dye Loading Buffer (1.2ml). (pg. 14)

SequaGel MD SSCP Kit EC-846 Contains SequaGel MD Monomer Solution (200ml) and SSCP Stop Solution (1.2ml).

Ammonium Persulfate EC-504 Exceeds ACS Standards. Low absorbed water results in consistent initiating capabilities. (pg. 32)

(pg. 14)

TEMED EC-503 Fractionally distilled twice to remove absolutely all inhibitory and fluorescent contaminants.

(pg. 33)

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Electrophoresis Applications - Gel Electrophoresis of DNA and RNA AquaPor LE

Applications

Electrophoresis

2.4 Agarose Gel Electrophoresis of DNA and RNA DNA and RNA strands are extremely large macromolecules. A 1 kilobase piece of single stranded DNA or RNA has a molecular weight of 330,000 daltons, larger than the vast majority of proteins. Often in the lab, genomic DNA fragments as large as 1000 kilobases (1 megabase) must be separated by gel electrophoresis. The separation of such large molecules requires an extremely open matrix structure. Agarose, which forms gels of sufficient strength at percentages as low as 0.5%, is the matrix of choice for separation of DNA or RNA over 1000 bp. Agarose is a natural polysaccharide, purified from seaweed (see Section 1.3.2 for a discussion of the agarose matrix). The crude precursor material (agar) has been used in some electrophoresis applications, but it contains a number of contaminants, which adversely affect the quality of the results. Sulfonated polysaccharides are the main problem, because they add strong negative charges to the gel matrix. As discussed in section 1.3.2, fixed charges on the matrix cause water to flow through the gel to balance the osmotic effects of the migration of their counter ions. This effect is known as electroendosmosis (EEO), which causes bands to smear or broaden. In addition, sulfonated polysaccharides can act as effective DNA mimics, profoundly inhibiting enzyme action in later processing steps, such as ligation or restriction analysis. To avoid these effects, agarose is purified to remove most of the endogenous contaminants found in agar. Gels prepared with agarose have low EEO, and thus excellent band resolution. Bands isolated from agarose gels can often be processed with enzymes without further purification, although this is not always the case. The 3-dimensional structure of an agarose gel is held together by hydrogen bonding. Because no covalent bonds link this network, the gel can be disrupted by heating. For this reason, agarose gels are easy to create and pour, one of the great advantages of this material. Agarose is simply melted into the proper volume of buffer, and the molten material is poured into a gel mold and allowed to cool. The buffer can be chosen to provide native or denaturing run conditions, so double stranded DNA, single stranded DNA and RNA can all be analyzed on agarose gels. Typically, agarose gels are run in a horizontal apparatus, with the gel lying under a thin layer of buffer (submarine gels). Agarose gels can also be run in a vertical format. This is generally done if discontinuous buffer systems or thin gels are required. Agarose electrophoresis is used for a variety of purposes. Specialty grades of agarose have been developed to fulfill specific requirements. The most commonly used variant is low-melting agarose, which has been modified to lower its melting temperature from over 90°C to around 65°C. This variation allows bands to be excised from a gel and then melted at a mild temperature to release the DNA. Unfortunately, low melt agarose generally produces gels which are difficult to work with because of low mechanical strength. However, National Diagnostics’ AquaPor LM, has exceptionally high strength for a low melt agarose, and produces gels which can be handled easily without breaking. Representing another popular form of agarose, National Diagnostics’ AquaPor ES has been modified for exceptional mechanical strength. This is particularly useful when extra large DNA fragments are run. The low percentage gels required to run megabase fragments of DNA are extremely flimsy. Extra strength agarose allows the use of very low percentage gels, permitting the analysis of even larger pieces of DNA. Finally, agarose can be refined to give matrices with enhanced resolution, such as National Diagnostics’ AquaPor HR. Standard agarose can achieve about 5% size resolution (resolved fragments must differ by at least 5%). High resolution agarose can resolve fragments differing by as little as 2%. This improvement allows the resolution of fragments below 500bp, and is ideal for analyzing PCR products. Another alternative for small fragments is National Diagnostics’ AquaPor 3:1, which is ideal for casting the high percentage gels necessary to sieve small DNA fragments. 62

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AquaPor 3:1

all around performance Gel %

Size Range (bp)

Gel %

2.0

200 - 3,000

1.75

250 - 4,000

1.5

300 - 5,000

1.0

400 - 12,000

0.75

1,000 - 23,000

AquaPor HR

highest resolution

AquaPor LM

small fragments

low melting point

Size Range (bp)

Gel %

50 - 1000

<500

100 - 1500

200 - 800

200 - 5000

300 - 3,000

400 - 10,000

500 - 20,000

0.75

1,000 - 20,000

0.75

600 - 25,000

AquaPor ES

high strength for PFGE

Gel %

Size Range (bp)

Gel %

Size Range (bp)

4.5

50 - 200

1.5

300 - 5,000

4.0

75 - 300

1.0

400 - 12,000

3.0

100 - 700

0.75

800 - 15,000

2.5

125 - 800

0.5

1,000 - 25,000

2.0

150 - 1000

0.3

5,000 - 50,000

Size Range (bp)

For a particular type of agarose, these tables indicate the percentage gel yielding the best separation over a particular range of fragment sizes.

2.4.1 Preparation of Agarose Gels

To determine the percentage gel to cast, consult the table above corresponding to the AquaPor agarose of choice. Protocol 2.4.1a Preparing an Agarose Gel Comb Selection: Use a thin (< 1 mm) comb with wide teeth for the sharpest, best-resolved bands.

Be certain the comb is cleaned scrupulously prior to use.

Buffer Selection:

Use 1X TBE for optimal resolution of DNA < 12 kb when the DNA will not be recovered.

Use 1X TAE for the best separation of DNA from 12 kb to 50 kb, or for DNA < 12 kb if the DNA will be recovered from the gel.

Use 1X Tris-Acetate (TAE without EDTA) if the DNA will be used for in-gel enzymatic processing.

Casting a gel:

Add buffer (at room temperature) to a flask that is 2.5 - 4 times the volume of gel solution. Add a teflon-coated stir bar.

Add AquaPor powder while stirring vigorously so the agarose is dispersed uniformly. Stir for 2 minutes to hydrate the agarose.

Tare the flask and solution.

Place in a microwave oven and heat at 100% power using 20-60 second intervals. Swirl gently between intervals to resuspend the agarose.

Continue the cycle of heating and swirling until the agarose is completely dissolved (no visible particles are present).

Add distilled water to return the solution to its initial weight and mix.

Cool the solution to 50-60°C before pouring the gel. Pour the solution into the mold so as to dispense the entire amount in 30-60 seconds, without generating bubbles. Insert comb.

After casting, chill the gel for 30 minutes prior to comb removal when using AquaPor LM, HR, and low (<1%) concentrations of AquaPor LE and ES. This will complete gelation, increase gel strength, and enhance DNA resolution.

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Preparation of Denaturing Agarose Gels

Several denaturants can be used with agarose. Alkaline gels are most often employed with single stranded DNA, because pouring and handling such gels is nonhazardous and convenient. However, because strong alkali will hydrolyze RNA, formaldehyde is used with RNA.

At high temperatures, alkaline conditions will hydrolyze the Agarose polysaccharide chains. To prepare an alkaline gel, the agarose is first melted in water and cooled to near gelling temperature. Buffer concentrate is then added and the gel is poured. Gel Preparation:

Dissolve 1.2g of Agarose in 98ml of deionized water. Stir agarose well into cold water, until all clumps are broken up. Heat the suspension in a microwave until it is a clear and homogeneous solution. Allow to cool to 15°C above gelling temperature.

Add 2ml of 50X Alkaline gel buffer.

50X Alkaline gel buffer: 1.5M NaOH 50 mM EDTA

Pour gel as described in Protocol 2.4.1a

Run gel in 1X Alkaline gel buffer. Sample Preparation: MIx Samples with an equal volume of: 60mM NaOH 2mM EDTA 20% Ficoll 0.06% Bromocresol Green

Restriction endonucleases are enzymes that cleave double stranded DNA at specific sites, generally 4, 6 or 8 base palindromic sequences. Because, in action, the enzymes are sequence specific, each piece of DNA has a recognizable pattern (or map) of restriction sites. This map serves as an easily identifiable “fingerprint” whereby the identity of a piece of DNA can be established without recourse to sequencing or blot hybridization. A map of restriction sites is also essential for planning subcloning experiments, in which a large piece of DNA is cut into smaller fragments for more convenient analysis.

Applications

Preparing Alkaline Agarose Gels

2.4.2 Restriction Digest Mapping

Electrophoresis

Protocol 2.4.1b

Applications of Agarose Gel Electrophoresis

To perform a restriction mapping experiment, 2-10µg of sample DNA is digested to completion in a set of separate reactions with 5-10 different restriction enzymes. These reactions provide the primary map information, giving the distances between the restriction sites and the ends of the DNA molecule, or revealing the existence of multiple sites for one enzyme. This information is extracted by running the digestion reactions on an agarose gel vs standards of known size, to determine the size of each restriction fragment generated. In the second stage of restriction mapping, the DNA is digested with pairs of enzymes (double digests) selected from the enzymes used in the single digests. The sizes of the fragments generated indicate the relative positions of the restriction sites for the two enzymes involved. Generally, a second round of double digests is then carried out to resolve remaining ambiguities in the map.

(Stock solution is: 2g Ficoll, 0.4ml 50X Alkaline buffer, 6mg Bromocresol Green, bring to 10 ml with deionized water.)

Protocol 2.4.1c Preparing Formaldehyde Agarose Gels (for RNA analysis) Note: RNA is subject to rapid degradation by RNase present in the environment. For optimal results, use water treated with DEPC.

Gel Preparation:

Melt 1.2g agarose in 87ml of DEPC water, by dispersing the agarose uniformly and heating in a microwave until all particles are dissolved.

Bring the melted agarose to 60°C.

Add 10ml 10X MOPS Buffer and 3ml 37% formaldehyde. FORMALDEHYDE IS VOLATILE AND TOXIC. WORK IN A HOOD FROM THIS POINT FORWARD.

10X MOPS: 0.2M MOPS pH 7 with NaOH 50mM sodium acetate 10mM EDTA Use DEPC treated water and RNase free reagents

Pour gel as in Protocol 2.4.1a. USE A FUME HOOD!

Allow gel to set for 1 hour.

Run gel in 1X MOPS Buffer

Sample Preparation: Sample Buffer: 65% formamide 22% formalin (37% formaldehyde) 13% 10X MOPS

Mix 40µl sample buffer with 10µl sample, heat to 55°C 15 minutes.

Add 10µl of: 50% glycerol, 1mM EDTA, 0.3% each bromophenol blue and xylene cyanol.

Figure 2.4.2a Restriction mapping involves treatment of a DNA fragment with restriction enzymes both singly and in combination. The electrophoresis of cleavage products yields a map of the DNA in terms of restriction sites.

Figure 2.4.2a Restriction mapping involves treatment of a DNA fragment with National Diagnostics AquaPor Agarose and Buffers restriction enzymes both singly and in combination. The electrophoresis of cleavage products yields a map of the DNA in terms of restriction sites. AquaPor LE EC-202 High quality, general purpose agarose ideal for most routine applications. Low EEO. DNase & RNase free. Unique low boil-over formulation. (pg. 16)

AquaPor 3:1 EC-206 Fine resolution of small DNA fragments. Yields strong gels with low UV background. Low viscosity makes pouring high% gels much easier. (pg. 17)

AquaPor LM EC-204 Low melting agarose. Certified for in-gel ligation and PCR. DNase & RNase free. Highest gel strength available in a low-melting agarose. (pg.16)

AquaPor HR EC-205 AquaPor HR combines high resolution with low melting. Resolves DNA down to 2% size difference (or 4 bp below 200 bp). DNase & RNase free. (pg. 17)

AquaPor ES EC-203 AquaPor ES is a premium, ultra high strength, ultra low EEO agarose. Ideal for Pulsed Field Gel Electrophoresis (PFGE). (pg. 17)

TBE 10X EC-860 Formulated with 18 MegOhm water. 0.2 micron filtration. More economical than bench-top buffers. (pg. 20)

TAE 50X EC-872 Ultra-Pure preformulated buffer for electrophoresis. TAE optimizes sample recovery with low melt agarose. (pg. 20)

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Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

2.4.3 DNA/RNA Purification from Agarose Gels

Applications

Electrophoresis

Low Melting Agarose

As with PAGE electrophoresis, the DNA or RNA resolved on an agarose gel is of high sequence purity, and it is often advantageous to recover this material. As later steps in processing may be sensitive to agarose, its contaminants or the components of the running buffer, it may be necessary to separate the nucleic acid from the matrix material. A variety of techniques have been developed to carry out such separations. In cases where agarose and buffer components will not interfere, low melting agarose gels are used for the electrophoretic separation. The gel is stained and the band(s) of interest excised. The excised material can be melted at 60-65°C, well below the melting temperature of any DNA longer than 30 bases. Processing is then carried out in the melted gel. (Section 2.4.4 details in gel enzymatic reactions.)

Glass Powder Elution

DNA purified from low melt agarose is sufficiently clean for many purposes. When further purification proves necessary, glass powder elution is an effective method. In this technique, the DNA is bound to finely powdered glass or microscopic glass beads in a high salt suspension. Agarose and other contaminants do not bind to the glass and can be washed away. The DNA is then eluted in water or a low salt buffer. A selection of glass powder elution kits are commercially available. DNA may also be purified from standard agarose gels by this technique, using sodium iodide (NaI) to disrupt the gel matrix.

Use of low melting agarose also facilitates purification from the matrix. The challenge of recovering DNA from a matrix arises from the fact that the nucleic acid molecules are caged in a 3-dimensional network of matrix molecules. Removing the DNA from this cage requires forcing the DNA through the matrix pores. Use of low melting agarose circumvents this problem by disrupting the matrix to release the DNA, simplifying the physical separation of the two components. Protocol 2.4.3a Figure 2.4.3a DNA Purification from agarose by electroelution

Purification of DNA from Low Melt Agarose Gels 1. Cast and run a gel (protocol 2.4.1a), stain (protocol 4.1.1b) and excise band(s). 2. Add 3 volumes TE buffer to the gel slice (for gels over 2%, use 5 volumes TE). 3. Melt agarose at 65° for 15-30 minutes. 4. Add to the melted agarose solution an equal volume of phenol, buffered to pH 8.0 with 0.1M Tris HCl and mix by vortex or vigorous shaking for 10 minutes - the longer it is mixed, the cleaner the final product. 5. Centrifuge in a microcentrifuge for 15 minutes at 10,000 rpm. 6. Collect upper aqueous phase. Do not recover any of the white interface material. (More DNA can be recovered by re-extracting the phenol/ interface with an equal volume of TE buffer, but this will also carry some agarose into the aqueous phase.) 7. Precipitate the aqueous fraction(s) with 0.1 volume of 3M sodium acetate and 3 volumes of cold ethanol. 8. Recover DNA by centrifugation, wash once with cold 70% ethanol and allow to air dry.

Figure 2.4.2a Restriction mapping involves treatment of a DNA fragment with Agarose and Related Products Useful in Purification restriction enzymes both singly and in combination. The electrophoresis of cleavage products yields a map of the DNA in terms of restriction sites. AquaPor LM EC-204 Low melting agarose. Certified for in-gel ligation and PCR. DNase & RNase free. Highest gel strength available in a low-melting agarose. (pg. 16)

AquaPor LE EC-202 High quality, general purpose agarose ideal for most routine applications. Low EEO. DNase & RNase free. Unique low boil-over formulation. (pg. 16)

TAE 50X EC-872 Ultra-Pure preformulated buffer for electrophoresis. TAE optimizes sample recovery with low melt agarose. (pg. 20)

TBE 10X EC-860 Formulated with 18 MegOhm water. 0.2 micron filtration. More economical than bench-top buffers. (pg. 20)

TE Buffer (100X) EC-862 100X Concentrated solution of 1M Tris-HCl, pH 8, with 100mM EDTA. 0.2 micron filtration.

AquaPor HR EC-205 AquaPor HR combines high resolution with low melting. Resolves DNA down to 2% size difference (or 4 bp below 200 bp). DNase & RNase free. (pg. 17)

(pg. 20)

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Electroelution

The most popular alternative to glass powder elution for the complete purification of DNA from agarose is electroelution. Because agarose gels are run in a horizontal apparatus, the gel can be manipulated during a pause in the run. This allows variations of electroelution to be performed that are not possible with vertical gels, which are encased in glass plates throughout the entire run. In the most straightforward form of electroelution, the band is excised from the gel and placed in a bag of dialysis membrane. This bag is then filled with electrophoresis buffer and placed in an electric field. The DNA migrates out of the gel slice and into the buffer, but it is too large to migrate out of the bag. Recovery is then just a matter of collecting the buffer from the bag. An alternative involves cutting a “trench” into the gel just ahead of the band of interest, and then continuing the electrophoresis until the band is eluted into the trench. Although such technique allows recovery of the band in a small volume of running buffer, it requires exact timing or running the gel on a UV transilluminator, to avoid running the band past the trench. Alternatively, instead of a trench, a slit can be cut in the gel just ahead of the band, and a piece of DEAE ion exchange paper can be inserted into the gel, so that the band is run onto the paper. The DNA binds tightly to the paper, and there is no need for exact timing and absolutely continuous monitoring of the run. Once all of the band is bound to the paper, recovery is accomplished by washing the paper in a high salt buffer. With this protocol it is often necessary to ethanol precipitate the DNA to remove the elution buffer.

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Electroelution into a Dialysis Bag 1. Cut the band of interest out of the gel, and trim away excess agarose. 2. Tie two knots in the end of a 1cm diameter dialysis tube, 5cm long. Alternatively, the bag can be closed with a plastic clip. 3. Place the band in the dialysis tube, add 0.5-1ml of TAE buffer, and seal the bag with knots or a second clip.

In Gel Restriction Digestion

5. Apply a voltage of 2-3 V per cm of distance between the electrodes. 6. Elute for 1 hr per kilobase of target length. For fragments over 5kb, elute overnight. 7. At the end of the run, reverse the polarity and run at 5V/cm for 1 minute to release any DNA adhered to the inside of the tubing. 8. Open the bag and recover the TAE. Rinse the bag with 0.5-1ml TAE and pool the TAE fractions. 9. Precipitate the DNA with 0.1 vol. 3M sodium Acetate and 3 vol. ethanol. Protocol 2.4.3d Electroelution into a Trough 1. Run the gel until the band of interest is adequately resolved. This is best done in an apparatus which can be mounted on a UV light box, running the gel in buffer containing Ethidium Bromide, and checking the band progress periodically. As an alternative, judge the run by the migration of the tracking dyes, staining the gel in Ethidium Bromide prior to elution to locate band of interest.

This technique is used when one DNA species from a complex mixture is to be digested. Without pre-purification, digestion would result in a complex mass of unidentifiable bands.

1. Run the DNA sample on a low melting gel of the appropriate concentration. Load sufficient DNA to provide 1-10µg of target material. 2. Stain the gel with ethidium bromide, and cut out the band(s) of interest. Do not expose the DNA to UV light for more than 1-2 minutes, or nicking and strand breaks may occur. 3. Weigh the gel slice, and determine the approximate volume by assuming 1mg = 1µl. 4. Heat the slice to 65°C in a sealed tube to prevent evaporation. Allow 5-10 minutes for the band to melt. 5. Equilibrate the melted band to 37°C, and add an equal volume of 2X enzyme buffer containing 5-10 units of enzyme per µg DNA in the slice. 6. Allow to digest for 1-2 hours at 37°C. 7. The digested DNA may be analyzed on a second gel, purified from the LM agarose solution (Section 2.4.3) or further processed as desired. Protocol 2.4.4b

2. Cut a trough 2mm wide just ahead of the band, 2mm wider than the band. Return the gel to the apparatus, and add or remove buffer until the top surface of the gel is barely above the level of the buffer. Fill the trough with buffer and run the gel until the band has entered the trough. If continuous monitoring is not feasible, stop the gel periodically to remove the buffer and refill the trough. An alternative, more reliable procedure is to cut a slit ahead of the band, and insert a piece of Whatman 3mm paper backed by a piece of dialysis membrane. Upon resumption of electrophoresis, the DNA is trapped against the dialysis membrane and can be easily recovered by eluting or centrifuging the buffer from the Whatman paper.

Protocol 2.4.3e Electroelution onto DEAE Paper Anion exchange paper will bind DNA tightly in the relatively low salt environment of an electrophoresis buffer. A strip of DEAE paper, placed in front of a DNA band, will effectively trap all of the DNA in the band. The DNA can be eluted in high purity with high salt. 1. Run an agarose gel and stain with ethidium bromide. 2. Locate band and cut slits in the gel just before and just after the band.

In Gel Ligation In most cases, the creation of cloned constructs requires the ligation of at least one, and more often two, fragments isolated from agarose gels. Isolation of bands prior to ligation prevents the generation of spurious constructs, and lowers the background of undigested vector DNA in the experiment. It is most convenient to perform ligation reactions directly in melted bands isolated from Low Melt agarose gels.

1. Excise the bands to be ligated from a low melting agarose gel of the appropriate concentration. Limit the UV exposure of the DNA to under 1 minute. 2. Combine the bands in a pre-weighed tube. Weigh the tube to determine the volume of agarose (assume 1mg=1µl). Melt the agarose at 65°C for 5-10 minutes, and bring to 37°C.

If the optimal ratio of the two fragments is known (i.e. 3:1 insert:vector for subcloning), the excised bands should be melted separately, and appropriate volumes combined to give the desired ratio.

3. Add an equal volume of 2X T4 ligase buffer, containing 1-2 units of T4 ligase. Mix well and ligate at 15°C overnight, or room temperature for 3-4 hours.

3. Insert a piece of DEAE filter paper into each slit, and return the gel to the electrophoresis chamber. 4. Continue to run the gel for 10-20 minutes, until the entire band is bound to the paper. The paper inserted above the band prevents any contamination from larger DNA fragments. 5. Recover the paper, and rinse briefly in electrophoresis buffer. Elute the DNA by placing the paper into 500 µl of 1M NaCl, and heating to 65°C for 30 minutes per kilobase of DNA. 6. Ethanol precipitate with 1ml of EtOH and wash pellet twice with 70% EtOH.

2.4.4 In Gel Enzyme Reactions

In many cases, the processing of DNA by enzymes is not impeded by agarose. Such reactions can be run directly in bands excised from low melting point agarose gels. The excised band is melted, mixed with the required buffer and enzyme, and then incubated at the optimal reaction temperature. The gel may solidify during the incubation without interfering with the reaction, and the agarose can then be remelted to recover the DNA.

Applications

Protocol 2.4.4a

4. Place the bag in a horizontal electrophoresis apparatus, and add 1X TAE until the bag is barely submerged.

Electrophoresis

NB: TAE, TBE and TTE, as well as most other common electrophoresis buffers, contain EDTA at 1-2mM. This level of EDTA is sufficient to inhibit most DNA modifying enzymes, by chelating the Mg2+ that these enzymes require. The effect of the EDTA can be avoided in two ways: more Mg2+ may be added to the reaction to saturate the EDTA, or less EDTA may be included in the electrophoresis buffer. Impure samples of DNA may be degraded by endogenous nucleases if the EDTA is decreased in the gel buffer, so if the purity of the sample is questionable, it is advisable to run the gel in the standard buffer and add supplementary Mg2+ to the subsequent reaction.

Protocol 2.4.3c

Protocol 2.4.4c In Gel PCR Amplification Although the PCR reaction is highly selective for the target DNA (Section 2.2.3) it is often beneficial to purify the template prior to amplification. In particular, if an amplification yields multiple bands, analysis of the individual bands requires separation on agarose, which is generally followed by re-amplification to provide sufficient material for analysis.

1. Run template DNA on a low melting agarose or high resolving gel of appropriate concentration. Stain with Ethidium Bromide and cut out the band of interest. Limit the time of exposure of the DNA to UV light to under 1 minute.

NB: Due to the extreme sensitivity of PCR, band contamination from adjoining lanes is highly probable. Use alternating lanes to minimize this problem.

2. Melt the DNA band at 65°C for 5-10 minutes. Add 1ml Nuclease free water for every 1µg of template DNA. Mix well. 3. 1µl of the above mixture will provide 1ng of template, which is sufficient for most amplification protocols.

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Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

Applications

Electrophoresis

2.4.5 Pulsed Field and Field Inversion Gel Electrophoresis (PFGE & FIGE) Standard agarose gels can resolve DNA fragments up to 75 kb, but analysis of genomic DNA often requires resolution of megabase (mb) fragments. Fragments greater in size than one megabase all run at the same rate on agarose gels (“limiting mobility”) and are not resolved from each other. This lack of resolution is because of the mechanism of sieving in agarose (or acrylamide) gels and the rodlike shape of double stranded DNA.

To migrate through a gel, DNA must pass through a large enough pore. The size of pore required depends on both the size of the DNA molecule, and its orientation relative to the pore. Molecules oriented perpendicular to their direction of migration present their full length to a gel pore, and thus require a very open gel structure to pass through. The longer a DNA molecule, the more angled it must be to pass through the pores of the gel. Limiting mobility is reached when a DNA molecule can only pass through the gel parallel to its direction of migration, presenting its end to each successive pore. This snakelike progression is called “reptation”, and requires that the DNA maintain itself in as straight a conformation as possible. This highly ordered state is thermodynamically unfavorable, and the DNA will “relax” into a less structured conformation rapidly when conditions permit. Once relaxed, a DNA molecule requires a finite time to reorient itself for further reptation. Longer DNA molecules require longer times to reorient. In PFGE & FIGE this difference in reorientation time serves as the basis of the electrophoretic separation of megabase sized DNA molecules. PFGE gels are run in a constantly changing electric field. Originally, gels were subjected to alternating voltage fields oriented at 90° to each other. Current protocols generally employ fields at 120° angles, and more complex systems use 3 or more angled voltages. In all cases, the effect is to force the DNA to continuously re-configure itself to migrate in a new direction. Larger molecules take longer to reorient and therefore make less overall progress through the gel. FIGE is a special case of PFGE, in which the fields are oriented at a 180° offset, directly opposing each other. In this case, the voltage pulses must be of different strength or duration, so that the DNA makes some net progress through the gel. In FIGE, the timing of the voltage pulses is critical, and must be matched to the reorientation times of the DNA of interest. If the reversing pulse is too short, larger DNA molecules will not reorient, while smaller molecules will reorient and begin to migrate backward. Upon resumption of the forward field, the larger DNA’s will be able to resume reptation, while the smaller pieces will rapidly reorient, but then have to make up the distance lost through reverse mobility. The result is that longer DNA’s will migrate faster than short pieces. Most often, a progressively longer series of pulses is used, to ensure good resolution over a wide range of sizes. FIGE is easier to use than PFGE, since it can be carried out in a standard horizontal gel apparatus. FIGE’s range of size resolution is more limited- up to 2mb versus 5mb for PFGE. A number of devices and systems for PFGE are commercially available. The gel running conditions must be optimized for the sample, gel apparatus and size range to be resolved. Parameters include overall pulse lengths, the ratio between forward and lateral pulse length, pulse voltages and the ramp rate between voltages. It is impractical to provide a general protocol which begins to address these variables. The user is referred to the instruction manuals provided with the PFGE units for suggested conditions for their own particular unit. One of the main challenges in PFGE experiments is to isolate intact DNA in the megabase size range. DNA of this length is easily sheared by turbulence in the solution. Shearing cleaves the DNA at random points, making it impossible to generate discrete bands during restriction digestion. A procedure has been developed in which the cells are lysed and the DNA released within an agarose block, which effectively protects the DNA from shearing forces.

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Protocol 2.4.5a Preparing High Molecular Weight DNA for PFGE & FIGE Electrophoresis 1. ENCASE THE CELLS IN AGAROSE BLOCKS

a. Prepare a mold to cast the blocks in:

Tape 1 end of a plexiglass mold, containing slots of the same size as the wells in the gel. Alternatives: i. Cast samples as dots of agarose on a glass or plastic surface, and cut to size. ii. Prepare a length of 2mm internal diameter Tygon tubing (~2cm/sample)

b. Prepare a solution of 1% agarose in lysis buffer:

10mM Tris HCl pH 8 100mM EDTA 20mM NaCl

c. Heat to melt the agarose and cool to 50°C.

d. Suspend the cells to be lysed in Lysis buffer at 108 cells/ml, and warm to 50°C.

e. Mix an equal volume of agarose and cell suspension, and pipette into molds (or draw up into tubing). Allow to set at 4°C. f. Recover the set plugs into 50ml centrifuge tubes. 2. LYSE THE CELLS a.

Incubate the agarose blocks in 50 volumes of lysis buffer containing 1% Sarkosyl detergent and 0.01% proteinase K, 16-24 hours at 50°C. Remove the supernatant, replace with fresh buffer/Sarkosyl/proteinase K mixture. Incubate an additional 16-24 hours at 50°C.

b. Remove lysis buffer, and wash 3 times at 50°C in 50 volumes of TE + 40 µg/ml PMSF, 1 hour each. (phenylmethyl sulfonyl/ chloride, a potent proteinase inhibitor)

Note: PMSF IS VOLATILE AND TOXIC. USE ONLY IN A FUME HOOD WITH ADEQUATE PRECAUTIONS. PMSF is inactivated after incubation at pH 9 or above for 1 hour.

3. DIGEST THE DNA a. Equilibrate the blocks in 10 volumes of 1X restriction buffer (optimal buffer for the desired enzyme). b. Remove the buffer and replace with 3 volumes of 1X restriction buffer containing 50 units of the appropriate enzyme. Incubate 16 hours at the digestion temperature.

c. Wash blocks for 1 hour in 50 volumes of TE @ 4°C.

4. The blocks may now be loaded directly into the PFGE wells.

Products for PFGE and FIGE AquaPor ES EC-203 AquaPor ES is a premium, ultra high strength, ultra low EEO agarose. Ideal for Pulsed Field Gel Electrophoresis (PFGE). (pg. 17) TE Buffer (100X) EC-862 100X Concentrated solution of 1M Tris-HCl, pH 8, with 100mM EDTA. 0.2 micron filtration.

(pg. 20)

TBE 10X EC-860 Formulated with 18 MegOhm water. 0.2 micron filtration. More economical than bench-top buffers. (pg. 20)

EDTA - ULTRA PURE EC-610 Molecular biology grade EDTA. Specifications include low insolubles (<0.005%) and >99% purity. (pg. 32)

Electrophoresis Applications - Gel Electrophoresis of DNA and RNA

2.4.6 RNA Electrophoresis

Preparation of RNA Samples

Isolation of intact RNA from cells depends upon the rapid inactivation of the endogenous RNase released when the cells are disrupted. The protocol below—based on the system of Chomczynski and Sacchi—uses the chaotropic agent Guanidinium Isothiocyanate (GTC) to disrupt the cells and dissolve cellular protein. RNase is inactive when dissolved in GTC in combination with reducing agents, so the disrupted cellular suspension preserves the RNA intact. The solution is then phenol extracted to remove the RNase. This extraction is carried out at a pH of 4.5, at which DNA partitions into the organic phase and is removed with the RNase and other proteins. Finally, the RNA is precipitated by ethanol and collected by centrifugation. Protocol 2.4.6a Guanidinium Isothiocyanate Isolation of RNA 1. Dissolve tissue in 10 volumes extraction solution. Tissue may be powdered under liquid N2 or homogenized into the buffer. Cells grown in a monolayer may be scraped into 1ml extraction solution/30cm2 growth area. Extraction Solution: 4M guanidinium isothiocyanate 25mM sodium Citrate, pH 7.0 0.5% Sarkosyl detergent 100mM 2-mercaptoethanol (use DEPC water for all stock solutions) 2. To 10ml of cells in extraction solution, add: 1ml 2M sodium acetate, pH 4.5 10ml buffer saturated phenol, pH 8 2ml chloroform: isoamyl alcohol, 24:1 Mix well between each addition by inverting tube several times. Mix complete solution 1 minute. 3. Chill solution to 4°C and centrifuge at 10,000g for 20 minutes at 4°C. 4. Recover aqueous (upper) phase and precipitate with 3 volumes of icecold Ethanol. Allow solution to precipitate at -20°C for 2 hours. 5. Collect RNA by centrifugation at 10,000g for 30 minutes at 4°C. Recover pellet. DO NOT ALLOW PELLET TO DRY OR IT WILL NOT REDISSOLVE. 6. If desired, redissolve pellet in 0.5ml of extraction solution and repeat steps 2-5. This gives a cleaner product but with some decrease in recovery. 7. Wash pellet with cold 70% ethanol. 8. Air dry pellet and redissolve in DEPC water. 9. Check recovery and purity - measure A260 and A280. An A260 of 1 indicates 40µg RNA/ml in the cuvette. Pure RNA has an absorbance ratio of A260/ A280 of 2.0. 1.8 or better is acceptable purity for Northern Analysis or cDNA Synthesis. A ratio below 1.8 indicates a need for further purification.

Agarose electrophoresis of RNA requires the inclusion of denaturing agents in the gel to disrupt secondary structures and ensure the relationship between molecular weight and mobility. Urea—used as a denaturant in polyacrylamide gels—disrupts the hydrogen bonds which hold the agarose gel together, and alkaline conditions—used in denaturing DNA electrophoresis in agarose—hydrolyze RNA. All denaturants which could be used for RNA analysis are toxic to some extent. Methylmercuric hydroxide (MMH) reacts reversibly with amino groups on RNA, and is an effective denaturant. However, its toxicity and high volatility make its use inconvenient and hazardous. Aldehydes also react with RNA to disrupt base pairing, and are somewhat safer than MMH. Protocols are given below for using formaldehyde or glyoxal.

Applications

RNases are small thermostable enzymes found throughout nature. In particular, RNases are found on the surface of human skin, where they are thought to play a role in defense against retroviruses. It is therefore of paramount importance to wear gloves throughout any RNA experiment, and that any glass or plasticware possibly touched with bare hands be treated prior to use. The use of certified RNase free glassware or disposable plasticware is recommended. RNases are extremely stable enzymes. Although denatured with boiling the enzymes renature upon cooling. Therefore, heating is not an effective method for eliminating RNase from solutions. Dry heating glassware is effective, and glassware may be rendered RNase free by heating to 250°C for 4 hours. Solutions which must be rendered RNase free may be treated in several ways. The most common approach is to use DEPC (Diethylpyrocarbamate). DEPC irreversibly inhibits RNase, and may then be removed by autoclaving. (NOTE: DEPC IS HIGHLY TOXIC AND VOLATILE. IT MUST BE USED ONLY IN A FUME HOOD). The limitations of DEPC are that solutions must be heated to remove the DEPC, (which would otherwise covalently modify the RNA) and that DEPC reacts with amines. Thus, to decontaminate heat labile, RNA containing or Tris buffered solutions another method must be used. Samples containing RNA are often decontaminated during extraction by treating with Guanidinium salts. Small volumes of Tris buffers are protected by adding RNase inhibitors: RNasin, a 40 kb protein or Vanadyl Ribonucleosides, which are transition state analogs which bind to and inhibit RNase.

Gel Electrophoresis of RNA

Electrophoresis

Electrophoresis of RNA presents unique challenges. Though RNA is isolated in single stranded form without complementary sequences, it must be fully denatured in order to obtain fractionation based on size. RNA molecules form complex and often very stable secondary structures, which are more difficult to denature than DNA. Additionally, RNA is extremely vulnerable to degradation by RNase enzymes found either in the sample or in the process environment. Effective procedures have been developed for the isolation of intact RNA and its analysis on denaturing agarose gels. Avoiding RNase contamination is critical to the prodcedure effectiveness.

Protocol 2.4.6b Formaldehyde Denatured RNA Gels 1. Cast Gel: Dissolve 1g agarose in 100ml of DI water. Heat to completely dissolve agarose crystals, and cool to 60°C.

Add 12 ml 10X MOPS, and 3.5 ml of 37% Formaldehyde.

10X MOPS: 0.2M MOPS acetate pH 7.0 0.05M sodium acetate 10mM EDTA Mix well and pour gel. Insert comb and allow to set for 30-60 minutes. 2. Sample Preparation: Mix: 4.5 µl of RNA (containing 10-20µg RNA) 2µl 10X MOPS 3.5µl 37% formaldehyde 10µl formamide Incubate at 55°C for 15 minutes. Add 2µl Loading Buffer: 50% glycerol 1mM EDTA 0.25% each bromophenol blue and xylene cyanol 3. Running the Gel: Run in 1X MOPS buffer at 10-20 V/cm for 2-3 hours, until bromophenol blue is 80-90% through the gel. Recirculate MOPS buffer to prevent pH drift. Use a peristaltic pump, or stop the run every 30 minutes and transfer buffer from cathode to anode and back. Protocol 2.4.6c Glyoxal Denatured RNA Gels 1. Cast Gel Dissolve 1.2g agarose in 100ml of 10mM sodium phosphate, pH 6.9. Heat to completely dissolve agarose crystals, and cool to 60°C. Inhibit RNases by adding sodium iodoacetate to 10mM. Mix well and pour gel. Insert comb and allow to set for 30-60 minutes. 2. Sample Preparation: Dissolve 10µg of RNA in 5µl of DEPC treated water. Add 6µl 6M glyoxal, 15µl DMSO, and 3µl of 0.1M sodium phosphate, pH 6.9.

Note: Glyoxal must be deionized before use. After deionization, the pH of the solution should be >5.

Incubate at 50°C for 1 hour. Add 5µl Loading Buffer: 50% glycerol 10mM sodium phosphate, pH6.9 1mM EDTA 0.25% each bromophenol blue & xylene cyanol

3. Running the Gel: Run in 10mM sodium phosphate, pH 6.9 at 3-5 V/cm for 3-6 hours, until bromophenol blue is 60-80% through the gel. Recirculate the buffer to prevent pH drift. Use a peristaltic pump, and magnetic stir bars in the buffer chambers. If recirculation is insufficient, the pH of the buffer will rise to the point that the glyoxal will dissociate from the RNA. USA: 1-800-526-3867 EUROPE: 441 482 646022

Applications

Electrophoresis

Electrophoresis Applications - Gel Electrophoresis of Proteins

Gel Electrophoresis of Proteins

3.1 DENATURING PROTEIN ELECTROPHORESIS - SDS PAGE

3.3 2-DIMENSIONAL PROTEIN ELECTROPHORESIS

Overview / Sample Preparation / Gel Preparation / Molecular Weight Determination / Peptide Mapping / Protein Purification / Generation of Antibodies

Isoelectric focusing

3.2 NATIVE PROTEIN PAGE

Overview / Sample Preparation / Gel Preparation / Activity Stains / Immuno Electrophoresis

Protein Electrophoresis...The Express Lane

roteins, unlike nucleic acids, present the researcher with a variety of chemical characteristics. Proteins fold into complex secondary, tertiary & quaternary structures. Their surfaces may be hydrophobic (membrane proteins) or hydrophilic, with greater or lesser distributions of charge and reactive groups. Electrophoresis techniques have been developed to take advantage of many of these characteristics, separating proteins on the basis of size, subunit composition, charge/mass ratio, isoelectric point, or combinations thereof. The vast majority of these techniques are carried out in polyacrylamide gels, which have pore sizes well matched to the usual range of protein molecular weights, generally from 5 - 150 kDa. Larger complexes are separated on high percentage (24%) agarose gels.

Separations are generally carried out in a vertical slab gel apparatus (see Section 1.5.2). This system allows the use of discontinuous buffers (which sharpen bands), more precise control of electrical parameters, and the exclusion of O2 from the polymerizing gel, which is required for polyacrylamide applications. Vertical gels are also thinner than horizontal gels of similar well capacity, which reduces staining times, often an issue in protein detection protocols. Vertical gels are poured between two glass plates, one of which is 1-2 cm shorter than the other. The plates are separated by spacers which determine gel thickness. A comb is inserted into the top of the gel mold after filling, to form sample wells. 0.75-2mm thick gels are generally selected for routine use. Thicker gels sacrifice resolution, as run anisotropies are exaggerated across thick gels. Thinner gels give excellent resolution, but their mechanical fragility is a nuisance. After casting, gels are clamped into an apparatus which places the top and bottom of the gel in contact with upper & lower buffer chambers. Samples are loaded (after comb removal) and voltage is applied. After the run, the apparatus is disassembled and the gel processed for detection of protein bands (see Section 4.2). Gels can be run in “full size” (16 x 30 cm or larger) or “mini” (8 x 10 cm) formats. 68

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Electrophoresis Applications - Gel Electrophoresis of Proteins

3.1 Denaturing Protein Electro- phoresis: SDS-PAGE

CHES Sample Buffer: 1% CHES pH to 9.5 with NaOH (Store @ -20° C up to 6 months) 2% SDS 1% DTT 10% glycerol Homogenize samples in 5 - 15 volumes of CHES buffer in a dounce homogenizer @ RT, 25 - 50 strokes.

Applications

The most commonly used denaturant is sodium dodecyl sulfate (SDS). SDS is an amphipathic surfactant. It denatures proteins by binding to the protein chain with its hydrocarbon “tail”, exposing normally buried regions and “coating” the protein chain with surfactant molecules. The polar “head” group of SDS adds an additional benefit to the use of this denaturant. Proteins solubilized in SDS bind the detergent uniformly along their length to a level of 1.4g SDS/g protein. This creates a charge/ mass ratio which is consistent between proteins. For this reason, separation on a polyacrylamide gel in the presence of SDS occurs by mass alone. SDS PAGE offers a rapid and relatively accurate way to determine protein molecular weights. Masses determined by SDS-PAGE are usually accurate within 5 - 10%, although occasionally proteins may retain enough secondary structure or contain sufficient charged groups to migrate anomalously. Histones, which carry a strong intrinsic charge, are an example of this phenomenon.

This protocol is sufficient for tissue culture cells, fluid samples (ie serum or cerebrospinal fluid), bacteria and soft tissues. For more highly structured samples, use the following buffer:

Electrophoresis

In their native form, proteins fold into a variety of shapes, some compact, some elongated. The rate of migration of native proteins through a sieving medium is therefore more a reflection of their relative compactness, and less an accurate measure of molecular weight. Denaturing the proteins nullifies structural effects on mobility, allowing separation on a true charge/mass ratio basis. It also separates subunits in multimeric proteins, allowing analysis of large, complex aggregates.

before loading. If particulate is present, centrifuge samples 5 minutes at 14k RPM in a microcentrifuge, and load the gel.

Heat homogenate to 95°C for 5 minutes and allow to cool. (homogenized samples will contain substantial amounts of cellular debris, which must be removed by centrifugation to avoid clogged wells. Centrifuge samples @ 14K RPM in microcentrifuge, 15 minutes. Note: yeast and bacteria:may be encapsulated in a layer of lipopolysaccharide (LPS) which will require enzymatic digestion with Lysozyme or Zymolyase prior to homogenization.

Sample purification with the ProtoGel Sample Prep Kit: (page 28) Samples which contain interfering small molecules (polysaccharides, lipids etc.) can be purified quickly to achieve optimum electrophoresis results using the ProtoGel Sample Prep Kit. 1) Add 5μl Reagent A and 10μl Reagent B to 100μl sample 2) Incubate 20 minutes to precipitate proteins 3) Centrifuge to recover protein/precipitant complex 4) Wash complex with acetone and centrifuge 5) Wash protein pellet twice with 70% ethanol 6) Remove ethanol and allow protein pellet to dry 7) Sample is now ready to be added to loading buffer- note that the sample has been concentrated as well as purified.

Figure 3.1a: SDS is the most commonly used detergent in protein electrophoresis. Treatment with SDS creates a uniform charge to mass ratio between different proteins.

3.1.1 Sample Preparation for SDS-PAGE

SDS is a powerful detergent, which will solubilize many cells and tissues. This greatly facilitates sample preparation for SDS PAGE because most samples will be completely dissolved by heating to 95°C in loading buffer (detailed below). A somewhat stronger loading buffer, containing SDS and dithiothreitol (DTT) at a higher pH, can be used for the homogenization of more difficult samples. In general, the goal of sample preparation is to denature the proteins fully, to disrupt any disulfide bonds through reduction, and to dissolve any particles which would interfere with electrophoresis. Incomplete denaturation will not fully saturate the proteins with SDS and will lead to blurred bands or altered mobilities. Failure to dissolve sample particulate completely will result in clogging of the gel causing streaking from the well to the end of the gel. Disulfide reduction is often required to release subunits from multimeric proteins. In many instances, it is instructive to run samples with and without reduction, to demonstrate which bands are released by disulfide disruption. Protocol 3.1.1a Sample Preparation for SDS-PAGE Electrophoresis Standard 2X Sample Buffer: 0.5M Tris-HCl, pH 6.8 4.4% SDS 300mM mercaptoethanol 10mg/ml bromphenol blue

Mix sample with an equal volume of 2X sample buffer (For greater reproducibility, employ National Diagnostics Protein Loading Buffer Blue 2X (EC-886)). Bring to 95° C for 10 minutes, cool to room temperature continued

3.1.2 Gel Preparation - Denaturing Protein Gels Two categories of buffer systems are available for SDS PAGE: continuous and discontinuous. (see Section 1.4 for a full discussion) Continuous systems use the same buffer in both the gel and tank. While continuous gels are easy to prepare and give adequate resolution for some applications, bands tend to be broader and resolution consequently poorer in these gels. Discontinuous buffer systems employ different buffers for tank and gel, and often two different buffers within the gel, with a third buffer in the tank. Discontinuous systems concentrate, or “stack” the protein samples into a very narrow zone prior to separation, which results in improved band sharpness and resolution.

In the classic SDS PAGE system developed by Laemmli (Section 1.4.2) the gel is divided into an upper “stacking” gel of low percentage (i.e. large pore size) and low pH (6.8) and a resolving gel with a pH of 8.8 with much smaller pores. Both gels contain only Cl- as the mobile anion. The tank buffer has glycine as its anion, at a pH of 8.8. When electrophoresis begins, glycine enters the stacking gel, where it is converted to a zwitterionic form with zero net charge. The glycine front moves slowly through the stacking gel, lagging behind the strongly charged, smaller Cl- ions. As these two current carrying species separate, a region of low conductivity, with a consequent high voltage drop, is created between them. This zone (a Kohlrausch discontinuity) “sweeps” the proteins rapidly through the large pores of the stacking gel, collecting the sample and depositing it at the top of the resolving gel in a focused narrow band. When the Kohlrausch discontinuity enters the resolving gel, the increase in pH ionizes the glycine so that it runs faster, dissipating the discontinuity. This allows the proteins to unstack and separate through the small pore resolving gel.

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Electrophoresis Applications - Gel Electrophoresis of Proteins

Protocol 3.1.2a

Protocol 3.1.2b

1. Prepare resolving gel and stacking gel casting solutions. Table 3.1.2a gives the formulations for SDS-PAGE resolving gels from 6 - 16% as well as the formulation for the stacking gel.

Formulate enough resolving gel solution to fill the cassette and formulate 1/5 that amount of stacking gel solution. De-gas the solutions for optimum reproducibility. To de-gas, stir the solution under aspiration for 10 minutes at room temperature.

Applications

Electrophoresis

Casting a Discontinuous SDS-PAGE Gel (The Laemmli System)

Resolving Gel Formulation % gel 6%

10%

12%

15%

Size Range (kd)

METHOD 1 Volume of ProtoGel, ProtoGel Buffer to use

METHOD 2 Volume of ProtoGel, and reagents to use

ProtoGel: 20.0ml ProtoGel: 20.0ml 60-200 ProtoGel Buffer: 25.0ml 1.5 M Tris-HCl (pH 8.8): 25.0ml Deionized H2O: 53.9ml 10% SDS: 1.0ml Deionized H2O: 52.9ml ProtoGel: 26.7ml ProtoGel: 26.7ml 40-140 ProtoGel Buffer: 25.0ml 1.5 M Tris-HCl (pH 8.8): 25.0ml Deionized H2O: 47.2ml 10% SDS: 1.0ml Deionized H2O: 46.2ml ProtoGel: 33.3ml ProtoGel: 33.3ml 20-80 ProtoGel Buffer: 25.0ml 1.5 M Tris-HCl (pH 8.8): 25.0ml Deionized H2O: 40.6ml 10% SDS: 1.0ml Deionized H2O: 39.6ml ProtoGel: 40.0ml ProtoGel: 40.0ml 15-70 ProtoGel Buffer: 25.0ml 1.5 M Tris-HCl (pH 8.8): 25.0ml Deionized H2O: 33.9ml 10% SDS: 1.0ml Deionized H2O: 32.9ml ProtoGel: 50.0ml ProtoGel: 50.0ml 15-50 ProtoGel Buffer: 25.0ml 1.5 M Tris-HCl (pH 8.8): 25.0ml Deionized H2O: 23.9ml 10% SDS: 1.0ml Deionized H2O: 22.9ml

Stacking Gel Formulation

ProtoGel: 2.6ml ProtoGel: 2.6ml ProtoGel Stacking Buffer: 5.0ml 0.5 M Tris-HCl (pH 6.8): 5.0ml Deionized H2O: 12.2ml 10% SDS: 0.4ml Deionized H2O: 11.8ml

Casting Tris-Tricine Gels Discontinuous SDS-PAGE employing Tris-Glycine as the tank buffer, the Laemmli system as described in Protocol 3.1.2a above, resolves proteins down to about 15 kd. However, below this size, the proteins do not “destack” from the SDS micelles running through the gel with the buffer front. In order to resolve proteins in this size range, the Tris-Tricine system of Schagger and von Jagow (1987) was developed. An Alternative to the Schagger and von Jagow System Running Tris-Tricine Gels using National Diagnostics 10X Tris-Tricine-SDS Buffer (EC-869) With National Diagnostics Tris-Tricine-SDS, you can extend the range of SDS-PAGE to resolve smaller proteins with minimal alteration of protocol. To provide this level of convenience, National Diagnostics streamlined the original method of Shaggar and Von Jagow (Anal. Biochem 1987 166, 36879) by developing Tris-Tricine-SDS cathode tank buffer to be compatible with the standard Laemmli gel/buffer system. This combination resolves proteins as small as 5kD. The researcher simply substitutes National Diagnostics Tris-Tricine-SDS in the upper (cathode) tank, with no other changes from protocol 3.1.2a, to extend the resolution of their gels. The Original Schagger and von Jagow System Tricine gels are poured in the same manner as Tris-Glycine gels (Protocol 3.1.2a), but the stacking and resolving gels are poured using the same buffer concentrate: Buffer Concentrate:

3.0M Tris-HCl, pH8.5 0.3% SDS

Resolving gel:

17ml Buffer Concentrate 17ml ProtoGel 12ml H20 5ml Glycerol

Stacking gel:

3ml Buffer Concentrate 1.6ml ProtoGel 7.5ml H2O

Polymerize as described for discontinuous protein gels in Protocol 3.1.2a, and run with 0.2M Tris-HCl pH 8.9 in the lower chamber, and 0.1M Tris, 0.1M Tricine and 0.1% SDS in the upper chamber.

Table 3.1.2a 2. Pour the resolving gel:

Add 1.0ml of fresh 10% Ammonium Persulfate solution for every 100ml of casting solution. Swirl gently to mix. Add 0.1ml of TEMED for every 100ml of casting solution. Swirl gently to mix. Pour the solution into the gel cassette. Fill the cassette to a level which will allow the comb to be inserted with 5mm between the bottom of the wells and the top of the resolving gel. Overlay the gel with 1-2mm of water saturated n-butanol to exclude O2 and ensure a flat interface between the resolving and stacking gels. Allow the gel to polymerize for 30 minutes. A line will become visible at the top of the gel as it polymerizes.

3. Pour the stacking gel:

Rinse the butanol from the top of the gel with water, and drain the water by inverting the gel. Add 0.2 ml of 10% APS and 20 µl TEMED for every 20 ml of stacking gel solution and fill the top of the cassette with this mixture. Insert the comb until the teeth are 5mm from the resolving gel. The comb should rest so that the tops of the well dividers are level with the top of the short plate. This excludes oxygen while ensuring that the dividers will fully separate the wells. Allow the stacking gel to polymerize for 30-60 minutes. Run the gel in 1X Tris-Glycine SDS.

Products for Denaturing Protein Electrophoresis ProtoGel EC-890 30% solution of Acrylamide and BisAcrylamide, 37.5:1 ratio. Filtered, Deionized, and Stabilized.

Insite Markers EC-897 Combined visible markers for run-time orientation and unstained protein standards for fluorescent detection. (pg. 27)

Tris-Glycine-SDS (10X) EC-870 ProtoGel Resolving Buffer (4X) EC-892 ProtoGel Stacking Buffer (4X) EC-893 Formulated with 18 MegOhm water. 0.2 micron filtration. Used in combination with ProtoGel to produce clear, reproducible, SDS-PAGE gels. (pg. 18)

ProtoStain Blue EC-727 Easy-to-Use colloidal Coomassie stain. Detects as little as 1ng of protein per band.

(pg. 8)

Ammonium Persulfate EC-504 Exceeds ACS Standards. Low absorbed water results in consistent initiating capabilities. (pg. 32) TEMED EC-503 Fractionally distilled twice to remove absolutely all inhibitory and fluorescent contaminants.

(pg. 33)

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(pg. 21)

Protein Loading Buffer Blue 2X EC-886 Ready to use buffer solution for the preparation of samples for SDS-PAGE. (pg. 18)

Electrophoresis Applications - Gel Electrophoresis of Proteins

Gradient Gels

Lower Solutions Upper Solutions

ProtoGel ProtoGel Buffer Gel % (30% 37:1 (1.5M Tris-HCl, pH8.8, Monomer % Acrylamide:MBA) 0.4% SDS) (ml) (ml)

Deionized Water (ml)

Sucrose (g)

3.3

11.6

4.0

5.3

9.7

6.7

8.3

6.7

5.3

3.3

13.3

Applications

Multiple gel casting units are available, particularly for mini-gel systems. The solutions given below are sufficient for 1 gel, and can be scaled up as appropriate for multi-gel casters. Similarly, the protocol given is for casting one gradient gel. For multi-gel systems, follow the instructions provided with the unit.

Gradient Gel Component Solutions

Electrophoresis

Gradient gels are cast with a higher concentration of acrylamide at the bottom than the top. The applications of gradient gels include the determination of protein molecular weights and the separation of molecules which co-migrate on uniform gels. The use of these gels is described in Section 3.1.3. Casting of gradient gels requires a gradient forming apparatus, and is more labor intensive than casting uniform percentage gels. For these reasons, precast gradient gels have become very popular.

Table 3.1.2c 2. PREPARE THE CASTING APPARATUS a. Assemble the gel cassette. Seal the bottom per manufacturerâ&#x20AC;&#x2122;s instructions, or use a molten solution of 1% agarose, allowing it to penetrate the bottom of the cassette by capillary action to a depth of 1 - 2 mm.

Figure 3.1.2a A gradient maker. As the apparatus empties, the composition of the out-flowing solution becomes progressively closer to the contents of B.

Gradient Casting Apparatus

The gradient is formed with a gradient-maker. The gradient maker must be positioned above the gel cassette to encourage flow due to gravity, or it can be emptied through a peristaltic pump. Gradient makers consist of 2 containers, joined by a narrow connector at their bases, with one container (A) having also an additional outlet in its base. As liquid is drained from (A), it is replaced from (B) due to the equilibration of hydrodynamic pressure which keeps the levels in (A) and (B) equal. (A) is constantly stirred, which causes the solution draining from (A) to be progressively diluted with (B) until, when the gradient maker is emptied, the outflowing material is essentially 100% B. Various shapes of gradients can be generated by varying the geometry of the system. In casting gradient gels, acrylamide monomer solutions are placed in the gradient maker, corresponding to the highest and lowest acrylamide percentages desired in the gel. Table 3.1.2b below gives suggested ranges of percentages for various protein size ranges. Table 3.1.2c gives formulations to produce the high and low percentage solutions needed to generate these gels. Sucrose is added to the high percentage gel solution to create a density gradient, which stabilizes the gel while it polymerizes, allowing more reproducible gradients between gels.

Protocol 3.1.2c

Pouring a Gradient Gel 1. PREPARE THE HIGH AND LOW PERCENTAGE GEL SOLUTIONS

Table 3.1.2b gives suggested gel compositions for various ranges of protein size. Table 3.1.2c gives formulations for the high and low percentage solutions to make these gels. Each solution will account for 1/2 of the total gel volume. To prepare a gradient which fully covers the desired range, both solutions must be completely consumed. The high percentage solution is subject to polymerization after APS addition, even in the absence of TEMED. For this reason, do not add APS until ready to pour the gel. Do not de-gas the solutions (de-gassing removes polymerization inhibiting O2) and keep the solutions cold prior to APS addition.

b. Place gradient maker on a stirring stand so that the bottom of the chambers are higher than the top of the gel cassette. Place a stir bar in each chamber. Attach a narrow bore tygon tube to the outflow. Attach a pipette tip to the other end of the tube, and clamp into position so that outflow is directed into the cassette from the top center. Angle cassette slightly to allow the solution to run down the tall plate. c. Close the stopcock between the gradient maker chambers and the outlet from the gradient maker. Place the low % gel solution into the non-outlet (reservoir) side of the gradient maker. Open the stopcock between the two chambers and allow 0.1 - 0.3 ml of solution to flow through to clear any bubbles. d. Place the high % gel solution into the outlet side (mixing chamber) of the gradient chamber. The next steps must be carried out rapidly, to avoid polymerization of the solutions before the gel is fully cast. e. Start mixing in the gradient maker. 3. CAST THE GEL a. Add the specified amounts of APS & TEMED to each chamber. (Per 100ml of casting solution, add 1.0 ml of 10% APS and 0.1 ml TEMED.) b. Open the stopcock between chambers. Some backflow into the low % reservoir may occur, due to the density variation between the solutions. It will not substantially alter the final gradient. Open the outlet, at a flow rate to drain the solutions in 5 - 8 minutes. Faster flow will cause a turbulence in the gel which will disrupt the gradient. Slower flow rates allow polymerization to occur before pouring is complete. c. When all of the solution is dispensed, remove the pipette tip from the top of the cassette. Overlay the gel with water saturated n-Butanol. Flush the gradient apparatus immediately with water to prevent polymerization within the system. d. After 1 hour, cast a stacking gel as detailed in Protocol 3.1.2a.

Separation Ranges of Gradient Gels Gel %

Size Range (kDa)

5-15

20-200

5-20

10-200

8-15

10-100

8-20

8-150

10-20

6-150

Notes: A peristaltic pump may be used to regulate the flow from the gradient maker. Gradient gels without stacking gels may be stored up to 1 week. Multiple gel casters are available from many manufacturers. Specific protocols optimal for each system are provided with the equipment.

Table 3.1.2b

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Electrophoresis Applications - Gel Electrophoresis of Proteins

Applications of Denaturing Protein Electrophoresis

Applications

Electrophoresis

3.1.3 Measuring Molecular Weight

The mobility (Rf) of a molecule in gel electrophoresis is determined by its free solution mobility, Y0 (= mobility in a gel of zero %) and the sieving action of the gel matrix. In denaturing protein electrophoresis, the addition of SDS to the electrophoresis buffer uniformly coats the proteins with negative charges, equalizing the charge to mass ratio for all proteins, thus making Y0 the same for all species. In this case, relative mobilities are determined solely by the sieving action of the gel. This sieving action is proportional to the molecular weight (MW) of the particular protein. Theoretical treatments suggest that log(Rf) should vary with MW, but most users use an empirical plot of log(MW) vs Rf for several standards of known MW to determine the MWs of unknowns. In practice the proportionality of log(MW) vs Rf holds true for most proteins, provided they are fully denatured, and provided the gel percentage has been chosen to match the molecular weight range of the sample. In fact, the actual plot of log(MW) vs Rf is sigmoidal (Figure 3.1.3a), because at high MW, the sieving affect of the matrix is so large that molecules are unable to penetrate the gel, while at low MW, the sieving effect is negligible, and proteins migrate almost at their free mobility, which in SDS is independent of MW. Given an appropriate selection of gel % (see Table 3.1.2a) and a protein which displays near-ideal behavior, molecular weights can be determined to within 5 - 10%. Molecular weights of non-ideal proteins can be determined by the use of Ferguson plots, discussed in Section 3.2.

3.1.4 Peptide Mapping

Peptide mapping involves controlled cleavage of a pure protein with small amounts of a pure protease to generate peptides of characteristic, reproducible sizes. These peptides can be separated on PAGE to produce a “fingerprint” characteristic of the protein. Peptide mapping can map cleavage sites in an unknown protein, or it can identify an unknown protein based upon its fingerprint identity with a previously tested sample. The polyacrylamide gel used can be either denaturing or non-denaturing, but SDS PAGE is most often used because it gives molecular weight information about the peptides produced. Small amounts of protease are used, so that minor variations in time and temperature of incubations will not overly perturb the results. Proteins for peptide mapping can be taken from bands sliced out of electrophoresis gels, or purified by standard means. Protocols are provided for the mapping of a pure protein and a protein in an acrylamide gel. Proteases for Use in Peptide Mapping Final Conc. (µg/ml)

Protease

pH optimum

Specificity

Trypsin

5-20

7-9

Arg, Lys

Papain

10-20

6-7

Arg,Lys,Gln,His,Gly,Tyr

Chymostrypsin

50-100

7-8

Aromatic

Elastase

50-100

7-9

Uncharged Aliphatic

Staph. Aureus ProteaseV8

50-100

4-8

Asp, Glu

Table 3.1.4a Protocol 3.1.4a

Figure 3.1.3a Although the overall graph of logMW vs. Rf is sigmoidal, it is nearly linear for a range of molecular weights depending on the percentage monomer of the gel.

Peptide Mapping - Purified Protein 1. Dissolve protein to 0.5mg/ml in digestion buffer; heat to 100°C for 2 min. Digestion Buffer: 0.125M Tris HCl pH 6.8 0.5% SDS 10% glycerol 0.0001 % Bromphenol blue (BPB) 2. Cool to 37°C and digest with protease for 30 minutes (see Table 3.1.4a for enzymes and amounts).

Gradient SDS Gels

If a gradient of acrylamide concentration is introduced into SDS PAGE, larger ranges of proteins may be analyzed on the same gels, with greater resolution. The complexity of the relationship between migration and molecular weight is dependant upon the shape of the gradient. The overall equation is of the form log(MW) α log(P), where P is the concentration of Acrylamide at the band position. A graph of log(MW) vs log(P) is linear, and allows the determination of MW’s from a set of standard protein positions. For linear gradient gels, the percentage of acrylamide is proportional to the position in the gel, so log(MW) will be proportional to log(band position). Therefore, a graph of log(MW) vs log(Rf) for a set of standards will be linear, and Rf values for unknowns can then be converted to MW values. On a 3 - 30% gradient gel, a range of proteins which differ in MW by up to 100 fold can be resolved and MWs determined. (Figure 3.1.3b).

3. Stop digestion by adding SDS to 2% (1/10 vol of 20% SDS) and 2-Mercaptoethanol to 10% and heating to 100°C for 2 minutes. 4. Load 10 - 15 - 20 µl (5 -10 µg) on a 10 - 15% SDS PAGE gel for analysis. Protocol 3.1.4b Peptide Mapping - Protein in a Gel Slice 1. Stain and destain as quickly as possible to avoid acid hydrolysis artifacts. 2. Cut out band of interest (containing 1-10µg of protein) and rinse slice in cold deionized water. Cut slice to the width of a well, because it will be loaded into the analytical gel. 3. Soak slice in 1X stacking gel buffer (see Protocol 3.1.2a) and 1 mM EDTA. 4. Place slice at the bottom of a well in the analytical gel (use a spatula or loading tip to place slice)

NB: The analytical gel for this protocol must have a stacking gel of at least 3 cm, to allow a space for digestion to occur.

5. Overlay slice with stacking gel buffer + 20% glycerol. Figure 3.1.3b With gradient gels log MW and log Rf are directly proportional over a broad range of values.

6. Overlay this with 10 µl of stacking buffer + 10% glycerol + protease + 0.01% bromophenol blue 7. Run samples into stacking gel. When the tracking dye reaches the bottom of the stacking gel, turn off voltage for 30 minutes to allow digestion to occur. Then continue run as usual.

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Note that smaller amounts of radiolabeled protein can be analyzed by this method.

Electrophoresis Applications - Gel Electrophoresis of Proteins

Analytical gels for peptide mapping:

The high resolution achievable with SDS PAGE means that the bands of protein visualized on the gel are (usually) extremely pure. Although the amount of protein which can be fractionated and recovered from a gel is relatively small (10 - 50 mg on large scale preparative gels, < 50µg on analytical or mini gels), enough can be recovered for a variety of purposes including amino acid analysis, antibody inductions , peptide mapping, microsequencing, and mass spectroscopy analysis. The challenge in recovering proteins from gel excisions is to overcome one of the forces which allowed the proteins to be separated in the first place, the sieving nature of the gel matrix. Due to the close agreement between pore size and protein size, passive diffusion in and out of the gel is extremely slow. As a consequence, external force must be exerted on the protein molecules to cause them to migrate out of the matrix or the gel must be altered itself. In the most basic technique, simple elution or “crush and soak”, the gel is broken, cut, ground or otherwise reduced to pieces < 1mm3 in size. This multiplies the surface area of the gel while maintaining a constant volume. With a greatly increased surface area to volume ratio, the concentration gradient between protein in the gel and protein in the elution buffer is strong enough to produce a realistic effusion rate. Though mechanically simple, the crush and soak technique has traditionally produced low yields (10 - 60%) requiring long incubations. Radical improvement with crush and soak can be obtained using the National Diagnostics Insite System for protein visualization (EC-759). The Insite System is an in-gel fluorescent stain for proteins, providing excellent sensitivity (10ng) more quickly than any other detection method. The Insite stain runs with the proteins during electrophoresis. No staining step is required after electrophoresis (only a short destain in water). Because no methanol or acetic acid is employed, the proteins are not fixed to the gel. Proteins visualized with Insite may be recovered by crush and soak at >80% yield after 8 hours soaking time. Although more complex and requiring special equipment, electroelution is a rapid, efficient technique, which essentially electrophoreses the protein out of the gel slice into the surrounding buffer. Because proteins require little time to migrate the 1 - 2 mm width of a slice, electroelution is fast, and usually yields from 70 - 90% recovery. For all techniques, processing of the gel prior to extraction can have a profound effect on sample recovery. Most staining techniques involve fixing, or denaturing of the protein bands (Section 4.2.1). Fixed proteins are insoluble precipitates, difficult if not impossible to recover. Fixation of the desired protein can be avoided by use of National Diagnostics’ Insite for detection or by use of the guide strip technique (Section 4.2.2), in which a duplicate lane is run and stained to provide a template for band excision from the unstained lane. If large amounts of protein are present, bands may be visualized without staining, using UV light at 302nm or by precipitating the protein-SDS complex with potassium or by chilling to 4OC. If fixing is unavoidable, recoveries can usually be enhanced by including SDS or Urea in the elution buffer, to redissolve the protein aggregates.

Crush and Soak Method for Recovering Proteins fromSDS-PAGE Gels Note: Staining of proteins in SDS PAGE gels generally involves “fixing” the gel by denaturing the proteins with acid and/or organis solvents. This denaturation prevents recovery by the crush and soak method. To recover proteins from SDS-PAGE gels, use the guide strip technique (page 89), then use the protocol below on the excised, unstained gel fragment.

Macerate or cut gel slice into pieces less than 1mm in any dimension, in a microcentrifuge tube. The smaller the gel fragments, the faster and more complete the elution will be.

Applications

3.1.5 Protein Purification using Denaturing Electrophoresis

Protocol 3.1.5a

Electrophoresis

The choice of gel system for the analysis of peptide mapping is dictated by the anticipated results. If a wide range of peptide sizes is anticipated, a gradient gel may be required. For peptides over 7 kDa, standard Tris Glycine SDS PAGE gels give superior results. Small peptides will require strongly denaturing fixatives to avoid loss of signal during staining (Section 4.2.1). For extremely small peptides, analysis on native PAGE gels (Section 3.2) may be superior. In native protein PAGE, separation is based partly on charge to mass ratio. This can enable the resolution of peptides that would run too close to the SDS/dye front in SDS PAGE.

2. Add 50 microliters of desired buffer to the tube. Adjust the volume of buffer as needed, so that the gel slice constitutes no more than 25% of the total volume in the tube. The protein will equilibrate between the gel slice and the buffer, so the percentage of total volume made up of added buffer is the maximum percentage recovery that may be expected. 3. Soak at 4oC. Recovery increases with soaking time: For 50% recovery, soak for 2-3 hours. For >80% recovery, soak 8 hours or overnight. 4. Gel fragments can be removed by filtration or centrifugation, or the supernatant can be pipeted off of the fragments.

Protocol 3.1.5b MALDI-MS Analysis of Samples Recovered from SDS-PAGE Gels 1. If necessary, concentrate small amounts of eluted protein by ultrafiltration on a Millipore UltraFree microcentrifuge filter (or equivalent), until the protein concentration is at least 1-5 pmol/microliter. 2. For the most consistent results across the sample spot, use the “crushed crystal” technique of sample spotting: a. Make up a matrix solution of 50% Acetonitrile, 0.1% TrifluoroAcetic acid, saturated with Sinnapinic Acid (~40 mg/ml) b. Spot 1 microliter of this solution on the MALDI sample plate, and allow to dry. Crush the resulting crystals by covering them with a glass microscope slide, and applying gentle pressure with the eraser end of a pencil. Remove the slide and gently brush off any loose crystals with a kimwipe, or blow them off with a stream of dry compressed air. c. Mix one part protein sample with 4 parts of the above matrix solution, and spot 1 microliter of this mixture onto the crushed crystals. Allow the sample to dry, and proceed with MALDI-MS analysis.

Protocol 3.1.5c Purification of Proteins from Denaturing PAGE by Electroelution Electroelution in small volumes of low levels (<20mg) of protein requires specialized equipment. For these applications, it is recommended that the protocols provided with the equipment be used.

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Electrophoresis Applications - Gel Electrophoresis of Proteins

Applications

Electrophoresis

Protein Precipitation

Researchers in proteomics often find themselves caught between two opposing forces. On the one hand, manipulation and detailed analysis of individual proteins requires fairly pure protein, concentrated in a small volume. On the other hand, most protein purification procedures involve dilution, sometimes by a factor of 1000 fold or more. In addition, many protein samples contain multiple protein species, and are very dilute to start with. Dilution not only makes analysis difficult, it also destabilizes many proteins, due to surface denaturation. Thus, one of the most commonly encountered challenges in the proteomics lab is the efficient concentration of a dilute protein solution. Concentration of protein samples is not only useful in and of itselfmost concentration procedures also discriminate between proteins and small molecules such as buffers. Concentrating a protein sample thus also offers the opportunity to change the buffer composition. This becomes important for handling effluents from column purifications, in particular ion exchange columns. These columns are generally eluted with a salt gradient. This leaves the sample in a large volume of high salt buffer- concentration of the sample, if done properly, will leave the researcher with a small volume of highly concentrated protein in a buffer appropriate to further manipulations. Concentration of proteins is generally accomplished by filtration, dialysis, or precipitation. Each of these techniques has its own advantages and drawbacks. Dialysis, for example, can be made to serve as a means of concentrating a protein sample, by dialyzing against a high molecular weight polymer which cannot penetrate the membrane. Water will diffuse out of the bag in an attempt to equalize the water activity on either side. While this can be effective, it is extremely slow, and leads to the loss of peptides smaller than the cutoff of the membrane (typically around 12,000 daltons). Also, the polymers used are often polydisperse, and smaller molecules can diffuse into the sample, posing a problem later in processing.

Ultrafiltration, accelerated by gas pressure or centrifugation, is a popular method for concentrating protein solutions. This technique uses membranes of defined pore size, allowing only molecules below a set size to penetrate. Membranes are available with cutoffs as low as 3000 Da, allowing the retention and concentration of even fairly small proteins. Ultrafiltration devices which fit into microcentrifuges make concentration by this method rapid and efficient. However, the cost of such devices, losses of protein bound to the membrane, and the loss of smaller peptides limit the application of this technique to a subset of the total possible samples. Precipitation of proteins is a rapid, inexpensive and simple method of recovering proteins from dilute solutions. Proteins may be induced to aggregate into complexes of greater density than the surrounding solution by the addition of organic solvents, salts or other agents. The precipitating agent must be chosen with care to achieve the desired result: some solvents, for example, will fail to precipitate smaller proteins, while trichloroacetic acid (TCA) is harshly denaturing, and unsuitable for applications requiring downstream activity or native structure retention. An ideal precipitating agent would be efficient, in that it would bring down even small amounts of protein from dilute solutions. It would be comprehensive, precipitating all proteins from a given mixture, preserving their relative proportions by having similar percent recoveries for all. And it would be non-destructive to the proteins, preserving as much structure as possible for downstream investigations. A table comparing the advantages and disadvantages of the more commonly used protein precipitation methods is shown below.

Protein Precipitation Methods Speed

Recovery

Protein to Protein Variabiliy

Effect on Proteins

Comments

â&#x20AC;&#x153;Salting outâ&#x20AC;?, entropic hydrophobic interactions

Slow: 1-2 hours for reproducible results

Variable: 50-90% depending on protein mixture and starting concentration

High: Each protein has its own optimum salt concentration

Gentle: Stabilizes precipitated proteins

Simple and non-hazardous, but requires strict control of all variables

TCA (+/- Acetone, Deoxycholate or other denaturants)

Acidic denaturation

Rapid: Under 30 minutes

Very good: Often used for quantitative labeling studies

Minimal: Essentially universal

Harsh: Strong acid denaturation can modify structure or hydrolyze backbone

Destructive to protein structure, very robust in terms of recovery and reproducibility

Acetone/Ethanol

Solvents - van der Waals, electrostatic and dipole forces

Rapid: Under 30 minutes

Variable: Some smaller proteins may be lost

Moderate: Some hydrophobic proteins may be soluble

Moderate: May cause denaturation due to inversion of hydrophobic areas

Easy and fairly gentle but with variable results for small proteins in particular

Polyethylene Glycols/polymers

Reduction in the activity of water

Slow: Requires 1-2 hours for full equilibrium

Variable

High: Varies strongly with the hydrophobicity of proteins

Gentle: Does not denature most proteins

Optimum conditions must be worked out for each experimental system

National Diagnostics ND Protein Precipitation Kit

Co-precipitation

Rapid: Under 30 minutes

Excellent: Greater than 90% Minimal: Essentially universal in most cases

Moderate: Immunospecificity preserved, although not enzyme activity in many cases

Universal, gentle method

Precipitant

Mode of Action

Ammonium Sulfate

Table 1.1a

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Electrophoresis Applications - Gel Electrophoresis of Proteins Protocol 3.1.5d Protein Precipitation using the ND Protein Precipitation Kit

2. Add 1/10 volume Reagent B to sample. 3. Allow to precipitate for 20 minutes at room temperature. Precipitate is comprised of Reagent A:B complex along with trapped protein molecules.

The kit is not selective for a particular class of protein. Reagent A binds non-specifically to proteins and the ratio of recovered proteins should reflect the proportion in the original solution. It is possible that individual proteins precipitate with slightly different efficiencies but this has not been observed in testing.

What are the upper and lower concentration limits of protein that can be precipitated? The lower limit for reproducible recovery of BSA is 100ng at a concentration of 0.25 µg/ml. Recovering more than 50µg of protein in a single tube at 200 µg/ml may not be ideal because it becomes more difficult to redissolve the protein pellet at the end of the procedure. Above this concentration it may be helpful to divide the samples among several tubes or dilute the sample before precipitation. We are not aware of any commercially available kit which can recover below 2µg which matches the simplicity of the ND Protein Precipitation Kit, which does not involve centrifuge concentration.

Applications

1. Add 1/20 volume Reagent A to sample in a centrifuge tube and mix well.

Is the kit selective for membrane proteins, cytosolic proteins etc?

Electrophoresis

National Diagnostics Protein Precipitation Kit precipitates >99% of all proteins, even complex mixtures in dilute solution. Interfering salts and surfactants are left behind in the supernatant. The precipitants are removed with a rapid and gentle acetonitrile or acetone wash, allowing the concentrated proteins to be recovered in a small volume of whatever buffer is optimal for the next procedure.

ND Protein Precipitation Kit FAQ

What MW of proteins can be precipitated?

Intact proteins in the range of 10kD -200kD have been precipitated successfully as analyzed on SDS-PAGE gels.

Are there special instructions for 2D electrophoresis and Mass Spectrometry?

2D electrophoresis and mass spectrometry require the sample to be as contaminant-free as possible. The wash step is very important in this regard as it removes traces of the precipitation reagents that were used. For 2D electrophoresis and mass spec several washes (at least 2) may be necessary to ensure no contaminants remain with the pellet. Centrifuge after each wash and be careful not to dislodge the pellet.

Does the concentration of salt in the sample have an effect on the results? Combined Reagent A and Reagent B form a coprecipitate with the protein. 4. Collect precipitate by centrifugation and remove supernatant. The pellet will be large. 5. Completely disperse pellet in acetone to dissolve away precipitated A:B complex. The solution should appear clear to cloudy, depending on protein concentration, with no visible clumps. Undispersed clumps will trap impurities which will be carried over into the final isolate. 6. Collect proteins by centrifugation.

Most salts at concentrations used in biological laboratories will not affect the precipitation method. However, the surrounding solution can effect kit performance in a few instances. Very high salt concentrations e.g. a saturated solution of NaCl (5.5M) will make it difficult to collect the pellet formed by adding reagent A and B due to the high density of the solution. In this case it may be helpful to dilute the sample before starting the precipitation. Furthermore, salts with chaotropic anions (thiocyanate, iodide, perchlorate) will affect the performance of the kit. Solutions with these salts will cause a precipitate to form as soon as reagent A is added. Chaotropic cations (guanidine) do not have this effect. Guanidine thiocyanate will affect performance of the kit but guanidine HCl will not. Sodium iodide and sodium perchlorate will affect performance but sodium chloride will not.

Do nucleic acids co-precipitate with proteins?

Yes nucleic acids do precipitate to some extent with this kit. The kit cannot be used as a way to purify proteins away from nucleic acids, as some nucleic acid will co-precipitate.

Does the pH of the starting solution affect precipitation?

The kit has been tested on protein solutions between pH 6 and pH 8 and no difference was seen in the recovery.

The protocol gives a choice between acetone, acetonitrile and 70% ethanol for the wash steps. Which one is best?

The choice of washing solution will depend on the application. An acetonitrile wash may help the recovery of low molecular weight proteins while 70% ethanol may be the best wash where salt removal is of utmost importance. Different washes can also be done sequentially. The best washing procedure for your application may have to be determined empirically.

After a simple wash, the small protein pellet is ready to be redissolved in desired buffer. 7. To remove salts and surfactants, wash pellet with acetone, acetonitrile or 70% ethanol. This step may be repeated if desired for heavily contaminated samples, or for downstream applications requiring the highest purity proteins. Collect proteins by brief centrifugation if necessary. 8. Redissolve pellet in desired buffer.

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Applications

Electrophoresis

Electrophoresis Applications - Gel Electrophoresis of Proteins

3.2 Native Protein Electrophoresis

although the curve is actually sigmoid in shape. This type of analysis is more subject to artifacts than the Ferguson plot, but is easier to carry out.

Proteins run on PAGE without SDS separate due to their charge to mass ratio. While native (nondenaturing) PAGE does not provide direct measurement of molecular weight, the technique can provide useful information such as protein charge or subunit composition. Native PAGE also has the potential for separating proteins of identical molecular weight which cannot be resolved with SDS-PAGE. In addition, proteins on native PAGE usually retain their activity. This allows enzymes to be detected by sensitive and specific activity stains and delicate proteins to be resolved and recovered in a biologically active form. The interpretation of native gels is more complex than the interpretation of SDS-PAGE gels. Not only can differences in relative mobility reflect differences in charge and/or mass, but proteins may also have a pH at or above the pH of the buffer, in which case they will not migrate or will “retro-phorese” backward into the upper buffer chamber. The equation governing protein mobility in native gels is as follows:

Finally, native gradient gels may be analyzed with activity stains, which simplify the pattern by only visualizing enzyme activities of interest. This can be useful when purifying an enzyme from a mixture of isozymes, or when studying the expression or activity of enzyme families. It also allows for the isolation of electrophoretically pure, active enzyme. Used in conjunction with gradient gels, activity stains can provide molecular weight information about otherwise uncharacterized enzymes. Of course, the technique is limited to enzymes for which chromogenic or chemiluminescent assays exist. Often an assay can be adapted from existing techniques with little effort. The critical requirement is that light be produced or a colored compound be deposited in the gel in an insoluble form, either by the enzyme (positive stain) or in a reaction inhibited by the enzyme (negative stain).

log Rf = log (Yo) - KRT, where:

Rf - relative mobility, normalized to the dye front or some other standard. Y0 - relative mobility of the protein in the absence of any sieving matrix. KR - “retardation coefficient,” the extent to which the gel matrix affects mobility. T - % monomer of the gel matrix.

As mentioned in Section 3.1.3, in the presence of SDS all proteins have the same Y0 so that a simple relationship exists between Rf and KR at any given T. In other words, SDS treated proteins—having identical charge to mass ratio—migrate at the same speed in free solution under electric force. With such proteins, if the mechanical resistance exerted by the gel is known, the mobility can be determined. This mechanical resistance, KR, is directly related to molecular weight, so that a determination of KR allows calculation of molecular weight. In native gels, the situation is more complicated. Both Y0 and KR can vary between proteins. Y0 is related to the charge, while KR varies with the mass. Separating protein mixtures and protein standards on gels of varying percentages allows for both the charge and mass of the sample proteins to be determined. The graphic analysis used is known as the Ferguson plot. On the Ferguson plot, log Rf is graphed vs %T for a range of %T (the originators of this system covered 7 different % gels). By the above equation the graph should have a slope of –KR and a y-intercept of Y0. Comparison with standards of known charge and size allows the charge and molecular weight of the samples to be found (Figures 3.2a & 3.2b). slope = -KR

Figure 3.2b Ferguson plot showing three proteins of the same mass but different charge.

Native Gradient Gels

Another, less laborious way of simplifying the interpretation of native PAGE gels is to run a gradient gel. Native gradient gels are poured in the same manner as gradient SDS-PAGE gels (Section 3.1.2). As proteins migrate through the increasing acrylamide concentration, into regions of ever smaller pore sizes, their mobility decreases. Eventually, each protein reaches its “pore-limit” where it slows to a minimum migration rate, which is constant for all proteins at their pore limit. The band pattern is stabilized at this point, so that gradient Native gel. PAGE approaches an equilibrium system,in that beyond a certain run length only minimal changes occur in the gel pattern. Once proteins reach their pore limit their relative positions are a direct reflection of their molecular weight. In a linear gradient, log MW is proportional to log Rf over a wide range, 76

Samples to be run on native gels should be prepared in a way which minimizes denaturation of the proteins. Avoid heat, strong detergents, foaming and overdilution. In addition the activity of endogenous proteases must be minimized. Keeping the sample cold and including protease inhibitors will be helpful in this regard. A formulation for a cocktail of protease inhibitors with a broad spectrum of activity is given below: Protocol 3.2.1a Preparation of Protease Inhibitor Cocktail (100X) Protease Inhibitor cocktail (100X): 200µg/ml aprotinin and antipain 100µg/ml pepstatin A 25µg/ml leupeptin and chymostatin 15mM benzamidine Store at -70°C for up to 2 months.

Ferguson Plots

Figure 3.2a Ferguson plot showing three proteins of the same charge but different mass.

3.2.1 Sample Preparation - Native Protein Electrophoresis

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Cell and Tissue Disruption

Tissue culture cells and soft tissue such as liver can be prepared by homogenizing 10 - 15 strokes in a dounce homogenizer. The homogenizer produces micro-turbulent regions which tear cells apart, but are not sufficient to disrupt a strong collagen matrix. Some grinding also occurs, which allows soft tissues to be prepared. Buffer choice is dictated by the requirements of the protein of interest, although some general principles apply. Isotonic (100-150mM salt) buffers of pH 6.5-8.5 are best for most applications. Large deviations from this range may destabilize proteins and will also introduce artifacts into the electrophoresis results. Tris or phosphate buffers work well in this pH range. Bacteria and more structurally strong tissues can be disrupted by sonication. Ultrasonic waves produce rapidly alternating high and low pressure waves, disrupting cells by shear force and cavitation. This generates high local heat output, so the sonication must by carried out on ice, in short bursts separated by relatively long recovery times. Another important consideration with this procedure is that positioning the probe too close to the solution surface may cause foaming, which can extensively denature sample proteins. For tough materials the French Press—which forces the material to be disrupted through a narrow opening at extremely high pressure—is appropriate. This generates shear forces and pressure differentials which tear apart most biological samples. The cell can be kept cold and flow rates controlled to minimize foaming, making it a good method for recovery of active proteins. Other methods for disrupting cells to recover active proteins include enzymatic digestion, grinding in liquid N2, grinding in alumina, and disruption in a blender. The topic is reviewed well in “Protein Purification” by Robert K. Scopes.

Electrophoresis Applications - Gel Electrophoresis of Proteins

3.2.2 Gel Preparation - Native Protein Gels

The basic protocols for preparing Native PAGE gels are the same as those given for discontinuous SDS PAGE gels (Section 3.1.2), substituting non-SDS buffers for those containing SDS.

Formulate enough resolving gel solution to fill the cassette and formulate 1/5 that amount of stacking gel solution. De-gas the solutions for optimum reproducibility. Stir the solution under aspiration for 10 minutes at room temperature.

2. Pour the resolving gel:

1.5M Tris-HCl, pH 8.8 0.6M Tris-HCl, pH 6.8 1.92M glycine, 0.25M Tris, pH 8.3

Protocol 3.2.3a

Formulations for Native Protein Gels Gel %

Activity Staining for Horseradish Peroxidase

DI Resolving Protogel Gel Buffer Water (ml) (ml) (ml)

2.5

5.5

2.7

2.5

4.8

10%

3.3

2.5

4.2

12%

2.5

3.5

Add 0.1ml of fresh 10% 6.5 2.5 Stacking 1.25 ammonium persulfate (APS) (Stacking Gel Gel buffer) solution for every 10ml of casting solution. Swirl Table 3.2.2a gently to mix. Add 10µl of TEMED for every 10ml of casting solution. Swirl gently to mix. Pour the solution into the gel cassette. Fill the cassette to a level which will allow the comb to be inserted with 5mm between the bottom of the wells and the top of the resolving gel. Overlay the gel with 1-2mm of water saturated n-butanol to exclude O2 and ensure a flat interface between the resolving and stacking gels. Allow the gel to polymerize for 30 minutes. A line will become visible at the top of the gel as it polymerizes.

3. Pour the stacking gel:

Horseradish Peroxidase catalyzes the oxidation of a wide range of substrates, transferring electrons from these substrates to H2O2 to produce water. Diaminobenzidine (DAB), when oxidized by HRP, produces an insoluble brown precipitate. Gels soaked in H2O2 + DAB will show brown bands over sites of peroxidase activity.

Applications

1. Prepare resolving gel and stacking gel casting solutions. Table 3.2.2a gives the formulations for native resolving gels from 6 - 12% as well as the formulation for the stacking gel. Resolving gel buffer (4x): Stacking gel buffer (4x): Running (tank) buffer (10X):

Because activity stains are specific for a given enzyme or family of enzymes, it is not possible to present general protocols. Examples of some well defined, commonly used activity stains are given below to indicate the general principles and uses of activity staining.

Horseradish Peroxidase

Casting Native Protein Gels

3.2.3 Activity Stains

Electrophoresis

Protocol 3.2.2a

Applications of Native Protein Gels

Rinse the butanol from the top of the gel with water, and drain the water by inverting the gel. Add 0.1 ml of 10% APS and 10 µl TEMED for every 10 ml of stacking gel solution and fill the top of the cassette with this mixture. Insert the comb until the teeth are 5mm from the resolving gel. The comb should rest so that the tops of the well dividers are level with the top of the short plate. This excludes oxygen while ensuring that the dividers will fully separate the wells. Allow the stacking gel to polymerize for 30-60 minutes. Run the gel in 1X Tris-Glycine buffer (see above).

Native Gradient Gels

Native gradient gels use the same pouring apparatus and techniques as SDS PAGE gradient gels. Table 3.2.2b gives the formulations for the high and low percentage solutions for Native PAGE gradient gels. See Section 3.1.2 for instructions and guidelines for pouring these gels.

After electrophoresis, soak gels in a solution containing: 5mM H2O2 0.5mg/ml DAB (diaminobenzidine) 50mM potassium phosphate pH 7 NB: DAB IS A SUSPECTED CARCINOGEN, DECONTAMINATE BEFORE DISPOSAL Brown bands at peroxidase sites will begin to appear in 1 - 10 minutes, depending upon activity. Stop reaction by removing gel to potassium phosphate buffer without H2O2 and DAB.

This assay can be adapted to detect a number of other enzymes. If H2O2 is left out of the buffer, and peroxidase is added at 50 µg/ml, the system will detect H2O2. Inclusion of amino acids allows the detection of the H2O2 producing enzyme amino acid oxidase. Similarly, addition of an amine will permit detection of amine oxidase which also releases H2O2. Inclusion of both H2O2 and HRP in the assay mix will turn the entire gel brown, except where catalase is present. Catalase is detectable in such gels as an achromatic zone.

Nitro Blue Tetrazolium

Another versatile stain is built around the reduction of nitro blue tetrazolium (NBT) to an insoluble blue formazan (Figure 3.2.3a). Diaphorases which reduce NBT at the expense of NAD(P)H, can be detected by soaking gels in 50mM sodium phosphate, pH 7.8 + NBT + NAD(P) H. This reduction can also be accomplished by superoxide. O2- producing enzymes can thus be detected, and superoxide dismutase (SOD), which removes superoxide, may be detected as an achromatic zone on uniformly stained NBT/ O2- gels.

Native Gradient Gel Component Solutions

Lower Solutions Upper Solutions

ProtoGel Gel % (30% 37:1 Monomer % Acrylamide:MBA) (ml)

Buffer (1.5M Tris-HCl pH8.8) (ml)

Deionized Water (ml)

Sucrose (g)

2.6

12.4

4.0

5.3

9.7

6.7

8.3

6.7

5.3

3.3

13.3

Figure 3.2.3a

Nitro Blue Tetrazolium (NBT)

Other Enzymes and Substrates

Chromogenic substrates also exist for β-galactosidase (x-gal), phosphatase (BCIP) and proteases. Creative adaptation of these assays allows the detection of a wide range of enzymatic activities on native gels.

Table 3.2.2b USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Applications - Gel Electrophoresis of Proteins

Applications

Electrophoresis

3.2.4 Immuno-Electrophoresis / Immuno- Diffusion Antibodies are produced by the immune system in response to foreign macromolecules. Each antibody binds specifically to one feature (epitope) on one macromolecule (antigen). This allows the use of antibodies for the detection and quantitation of specific proteins in complex mixtures. Antibodies are generally isolated from animal serum, unless they are produced from tissue culture as monoclonals. The serum must be titrated to determine antibody concentration and specificity. Concentration determinations are often needed for antigen mixtures as well. Immunodiffusion and immunoelectrophoresis are useful techniques for these purposes. If antibody and antigen are present in solution at approximately equal concentrations, they form an aggregate, which precipitates. This precipitate can be dissolved by the addition of an excess of antibody or of antigen (Figure 3.2.4a).

Protocol 3.2.4a Radial Immuno-Diffusion 1. Melt 1g Agarose in 100ml PBS. 2. Spread 1ml of this solution on a glass plate or microscope slide. Allow to dry. This precoats the slide and allows the gel poured in the next step to adhere to the slide. 3. Pour enough of the agarose solution onto the precoated slide to cover it to a depth of 1 - 2 mm. Allow this to cool until gelled. 4. Punch wells in the gel with a 2 - 3 mm punch. The end of a 1ml glass pipette works well. 5. Place antigen and antibody solutions in adjacent wells and allow plate to diffuse 24 hours. It will be necessary to run multiple dilutions of sample and antibody to determine optimum concentrations of each.

Immuno-Electrophoresis

Agarose gels have a small number of fixed charges, which cause a phenomenon known as electroendosmosis (Section 1.3.2). EEO causes a slow net flow of water through the gel away from the positive electrode. At a pH of 8.5, antibodies are nearly uncharged, and their slow electrophoretic migration is nullified by the EEO flow through the agarose. Most other proteins are highly charged at pH 8.5, and will migrate through an agarose gel. If antiserum is included in the gel matrix, immuno precipitates will form, in a pattern dependent upon the antigen concentration. This technique, immuno-electrophoresis, is much faster than immuno-diffusion, and has been applied in a variety of geometries, to analyze simple or complex samples. Protocol 3.2.4b “Rocket” Immuno-Electrophoresis “Rocket” Immuno-Electrophoresis is used as a rapid way to quantitate antigen in complex samples. 1. Cast a 1% agarose gel, in buffer at pH 8.6 containing typically 1% of the desired antiserum.

Figure 3.2.4a When present at approximately equal concentration, antigen and antibody will precipitate as an aggregate. The figure above illustrates a principle underlying the usefulness of immunodiffusion, that with shifts in antibody concentration, a corresponding shift in the region of precipitation will occur.

2. Into wells punched at one end, load antigen solutions. Run gel with wells closest to the negative electrode.

In a gel matrix, if a gradient is established in which antibody concentration decreases linearly in a given direction, while antigen concentration increases in the same direction, a precipitate will form a band perpendicular to this direction, at the point of approximately equal concentration This is the basis of both immunodiffusion and immunoelectrophoresis. In both of these techniques, a gradient of antigen, or of antibody and antigen, establishes a line of precipitate in the gel. The position of the line is indicative of the concentration of antigen or antibody in the sample, and may be calibrated against standards. Figure 3.2.4b shows different variations of the immunodiffusion assay.

Ag Ab

Mixtures with multiple antigen species which cross react with the same antiserum may be analyzed by running them first on an analytical gel. Then cut a strip from that gel and lay it in a slit cut into the immuno-electrophoresis gel to form a large well. (Figure 3.2.4c). The resulting pattern shows the positions of strongly reacting antigen species.

3. Precipitates can be stained with Coomassie Blue R-250. Detection limits are usually sufficient to quantitate 0.1 - 0.2 g/ml of antigen.

c. Figure 3.2.4b Of the common patterns of wells containing antibody antiserum (Ab) and antigen samples (Ag) used in immunodiffusion, the system of parallel slots (a) can demonstrate the presence of antigen and roughly estimate concentration; the three-well pattern (b) can demonstrate the immunological identity (or not) of two antigen samples; radial immunodiffusion (c) introduces the capacity for comparative analysis unavailable with the three-well pattern.

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First dimension

Second dimension

As the antigen proteins enter the gel, they form a concentration gradient, which at some point gives the proper concentration for precipitation with the antibody in the gel. The more concentrated the antigen, the further it must run to be diluted to precipitable levels. The result is that each sample gives a “rocket”, the length of which is proportional to the concentration of antigen in the sample.

Figure 3.2.4c Crossed immunoelectrophoresis of antigens and antiserum. In the first dimension, proteins are separated by standard electrophoresis. The separated proteins are then run into the second dimension gel at an angle of 90° from the first dimension. The second dimension gel contains antiserum, generating an immunoprecipitate pattern.

Electrophoresis Applications - Gel Electrophoresis of Proteins

3.3 Two Dimensional Electrophoresis

Proteins carry charged groups on their surface (see Section 1.1.2 for a discussion of protein structure). Each of these functional groups has a pK, which corresponds to the pH at which half of the members of that group are protonated. Above the pK that group can be considered fully deprotonated, below the pK, fully protonated. Thus, as the pH changes, the net charge on a protein’s surface will change. At high pH, most proteins will have many deprotonated surface groups, and will carry a net negative charge. At low pH, with many protons added to the surface, most proteins have a net positive charge. At some intermediate pH, different for every protein, the net charge on the protein will be zero. It is important to remember that this is a net charge - the protein is not uncharged - it carries equal numbers of positive and negative charges. The pH at which a protein has a net charge of zero is designated its isoelectric point (pI). In isoelectric focusing (IEF) a pH gradient is established along the length of the gel. Under the influence of electric force, proteins migrate through this gradient until they reach the pH zone equal to their pI. The orientation of the voltage is chosen so that if a protein is in a region where the pH is above its pI it moves toward a lower pH zone. If the protein is in a pH below its pI, it has a positive charge and moves into higher pH regions. This gives rise to the “focusing” aspect of IEF, as proteins are continually swept back into tight bands centered on the appropriate pI (Figure 3.3.1a). IEF is thus an equilibrium electrophoresis system, run until protein movement ceases.

Ampholytes

Applications

3.3.1 Isoelectric Focusing

Isoelectric Focusing

Electrophoresis

Conventional electrophoresis techniques can separate up to 100 different proteins on one run. Typically, cell or tissue extracts contain thousands of proteins, most of which will not be resolved into single bands with separation based on any one parameter, such as size or net charge. For any one size range, there is a high probability of more than one protein falling into this range. Separation on the basis of two parameters, usually size and isoelectric point, lowers the probability that two proteins will overlap, and allows the resolution of thousands of protein species on one gel. Such two dimensional separations are carried out by running a one dimensional gel to separate by the first parameter, and then laying this gel or a lane cut from it, across the top of the second dimension gel, the first gel serving as the “sample” for the second. Typically, the second dimension gel is an SDS PAGE gel, because this gives a direct measurement of a protein’s size. The first dimension usually employs isoelectric focusing (IEP). IEP separates by the isoelectric point (pI) of the protein - the pH at which the protein sample will not migrate in an electric field, the protein’s net charge being zero. A two dimensional gel can give size and pI information on thousands of proteins in one run.

Various mixtures of amphoteric substances have been used as ampholytes: amino acids, proteins, and poly acidic poly basic synthetic molecules. Natural amino acids have poor conductivity and poor buffering capacity in their zwitterionic state, making them poor candidates. Proteins can be good ampholytes, but they interfere with analysis of the sample, by introducing new proteins into the mixture. Polycarboxylic acid polyamines are the most commonly used ampholytes. These molecules have excellent buffering capacity and conductivity across a broad pH range, and are usually provided in a molecular weight range of 300 - 500, which is small enough to avoid interference with most subsequent processing. Their sole disadvantage is that they may bind tightly to the proteins, due to ionic interactions, and can be very difficult to remove. Protocol 3.3.1a

IEF is frequently the first step in 2-dimensional electrophoresis. The apparatus best suited for this purpose is a “tube gel” system. The gels are cast and run in glass tubes with an internal diameter matched to the thickness of the second dimension gel. 1.5mm gels are commonly used. After the run, the IEF gel is extruded from the tube and laid across the top of the second dimension gel. This system is assumed in the protocol below. IEF gels can also be run as slabs, which allows an increased sample throughput. Slab IEF gels can be cut into strips for loading onto second dimension gels.

1. PREPARE THE GELS a. To formulate gel solution, dissolve 4g urea in 3 ml H2O + 1ml ProtoGel. Warm to 37°C if needed. De-gas for 10 minutes under aspiration. b. Add 150µl NP-40 detergent and 0.4ml ampholytes, mix and filter through a 0.22µm filter. c. Add 30µl of 10% APS, and 3µl of TEMED. Mix well and place solution in a 10ml syringe with a 22ga needle as long as the gel tubes. d. Insert needle into gel tube until it reaches the bottom, and fill tube, withdrawing needle as the fluid level rises. Use the needle to dislodge any bubbles. Allow the gels to polymerize for at least 2 hours. 2. PREPARE THE SAMPLES a. Add 15ml sample buffer/gram tissue: 9M Urea 4% NP-40 2% Ampholytes (pH 9-11) 2% Mercaptoethanol pH to >9 with NaOH. b. Homogenize if necessary. Incubate at room temperature for 10 min., then centrifuge for 1 hr. at 100,000g. Remove supernatant without disturbing pellet, which contains materials likely to clog the IEF gel. 3. RUN CONDITIONS a. Fill lower tank with 0.1% phosphoric acid. Place gels in the apparatus and fill the upper tank with 20mM NaOH. Use a syringe to dislodge any bubbles from inside gel tubes. Any bubbles in the tubes will distort the electric field and prevent gels from completely focusing during the run. b. Samples prepared as above can be loaded by layering onto the tops of the gels. Run at 500-700V (depending upon the apparatus used) for 16-24 hours. At the end of the run, mark the top end of each gel with a small amount of bromphenol blue.

Figure 3.3.1a In isoelectric focusing, a protein stops migrating when it enters the zone in which the surrounding pH equals its isoelectric point, pI.

The pH gradient in IEF gels is generated by the inclusion of a mixture of ampholytes of varying pI. Ampholytes are low molecular weight amphoteric molecules, which, like protein molecules, migrate through the gel until they reach a region where the pH equals their pI. Unlike the proteins, the ampholytes are present in high enough concentration to change their local pH. The gel is set up with a uniform mixture of ampholytes throughout, and its anodic and cathodic ends are immersed in dilute acid and base respectively. With the application of voltage, the ampholytes separate into zones of defined pH. If the ampholyte system is well designed, a smooth gradient of pH is created, with no abrupt charges, or “steps”. Commercial systems are available in broad range (2 - 12 pH) or narrow range (extending 2 pH units across the gel).

4. POST ELECTROPHORESIS

IEF gels may be coomassie stained (Section 4.2.2), but most often they are loaded onto SDS PAGE gels for second dimension analysis. Extrude the gels by applying pressure to one end of the tube with a pipette or syringe, while holding the other end over a vial or tray. Extruded gels may be frozen at -70°C for later analysis.

5. LOADING AN IEF GEL ONTO A SECOND DIMENSION SLAB a. Place the IEF gel in a solution of 20% glycerol, 4% SDS, and 250mM Tris-HCl, pH 6.8, with 1% mercaptoethanol added just prior to use. Incubate the gel in this solution for 5-10 minutes. b. Dissolve 0.1g agarose in 20ml of Tris-Glycine-SDS buffer. Heat until the agarose is melted. c. Overlay the second dimension gel with 1-2 mm of agarose, place the first dimension gel over the agarose, being careful not to trap any air bubbles. Overlay the gel with another 1-2mm of agarose solution, and run the second dimension gel.

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Applications

Electrophoresis

Electrophoresis Applications - Post Electrophoretic Analysis

Post Electrophoretic Analysis

4.1 DNA & RNA Detection

Fixing and Staining / Autoradiography /

Blotting

4.2 Protein Detection Fixing and Staining / Blotting

Post Electrophoretic Analysis...To See or Not to See!

lectrophoresis accomplishes the separation of a transparent sample of macromolecules across a clear or semi-clear gel matrix. Once this separation is achieved, further steps are required to detect the sample molecules within the gel. As samples routinely contain more than 100 separable components, each band may represent less than 1% of the total material loaded. The sensitivity of detection rapidly becomes a key issue. In addition, diffusion within the gel after the electric potential is removed causes band spreading, decreasing resolution over time. Speed and the ability to immobilize or â&#x20AC;&#x153;fixâ&#x20AC;? bands are important parameters to be considered. Finally, it is often necessary to determine the relative amounts of material detected in several samples loaded on a gel. Thus a wide linear response is desirable.

Detection methods may be broadly divided into two types: staining, in which a small molecule is selectively bound to the sample components, causing bands to become visible and/or fluoresce; and blotting, in which bands are detected on the basis of their specific interactions with macromolecular probes. The use of macromolecules as probes for detection requires that the bands be transferred out of the gel onto a solid support, a process known as blotting, which has given its name to an entire class of processes.

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Electrophoresis Applications - Post Electrophoretic Analysis

4.1 DNA and RNA Detection 4.1.1

Staining of Nucleic Acids

Applications

EtBr staining is strongly enhanced by the double stranded structure of native DNA. Staining of denatured, ssDNA or RNA is relatively insensitive, requiring some 10 fold more nucleic acid for equivalent detection. Another limitation is that the fluorescence of ethidium is quenched by polyacrylamide, reducing sensitivity by 10-20 fold in PAGE gels.

Electrophoresis

Nucleic acids are usually loaded onto gels at levels below 10 µg. For a typical Southern Blot (Section 4.1.2) a single copy gene will be present at <1 in 106, or <10pg/band for every 10 µg loaded. Alternatively, a restriction digest of 1 µg of a 6 kilobase plasmid which cuts the DNA into 6 unique pieces, the smallest of which is 600 bp, will present bands larger than 100 ng. A wide array of staining and blotting techniques have been developed to cover this range of detection, offering low sensitivity, great speed and convenience at one end and extreme (but labor intensive) sensitivity at the other. Some of the most sensitive techniques are destructive, rendering the DNA inactive and not suitable for recovery. Most of the less sensitive techniques preserve the DNA structure intact, and are commonly used where DNA is to be purified from the gel for further processing. In general, staining techniques aim for sensitivity in the nanogram range and recovery of intact DNA.

DNA, yielding low background and a detection limit of 1-5 ng/band. The major drawback is that it is a potent mutagen. Solutions must be handled with extreme caution and decontaminated prior to disposal. Nonetheless, the sensitivity, simplicity (the dye may be run in the gel with the DNA if desired, eliminating a separate staining/destaining process) and nondestructive nature of EtBr staining have made it the standard stain for double stranded DNA.

Protocol 4.1.1b Detection of DNA/RNA using Ethidium Bromide *CAUTION: ETHIDIUM BROMIDE IS A POTENT MUTAGEN. HANDLE ONLY WITH GLOVES AND PROPER PRECAUTIONS. Method I - Including ethidium bromide in the gel and buffer GEL PREPARATION

Figure 4.1.1a When ultraviolet light strikes a DNA molecule, it may be absorbed, transmitted, or, if a fluorescent dye is present, re-emitted as visible light.

1. Dissolve agarose in buffer as described in Section 2.4.1. 2. Allow gel to cool to 60-70°C. 3. Add EtBr to 0.5 µg/ml final concentration. (Stocks are generally 10 mg/ ml, and require 5µl stock/100ml gel). 4. Pour gel and allow to set as usual.

UV Shadowing

Detection of DNA/RNA in solution is usually done by measuring its UV absorbance at 260 nm. This absorbance is due to the ring systems in the nitrogen bases, and can also be used for low sensitivity detection of DNA/RNA in gels. In a technique known as UV shadowing, the gel is placed on plastic wrap over a UV fluorescent TLC plate. The dye in the plate is excited by placing a near-UV source over the gel. Dark areas are observed where DNA in the gel absorbs the UV light. The sensitivity of this method is limited, on the order of 10-50 µg, and its use is limited to thin gels, which do not excessively attenuate the UV light.

Nuclistain

National Diagnostics’ Nuclistain offers a significant increase in sensitivity over UV shadowing along with the convenience of visual staining. With Nuclistain there is no need for UV light. Nuclistain is a blue dye which binds to DNA, revealing blue bands after destaining with water. Nuclistain detects DNA down to levels of 50ng. Nuclistain does not modify DNA in any way and is easily removed from DNA following band isolation. Protocol 4.1.1a

BUFFER PREPARATION 1. Prepare enough buffer to fill the apparatus. 2. Add 5µl/100ml EtBr stock. RUN GEL Upon completion of run, place gel in plastic wrap on a UV light box. Bands will appear bright orange on a faint orange background. Notes: Gels and buffers must be decontaminated prior to disposal. See your local health and safety office for your institution’s preferred method. This method will detect ~ 5ng. of DNA. Destaining in water or 1 mM MgSO4 may be required to achieve full sensitivity. As an alternative, ethidium may be included in the gel, but not the buffer. Ethidium is positively charged, and will migrate in the opposite direction from the DNA. In general, sufficient ethidium will remain bound to the DNA even at the cathode end of the gel. Such gels will have an area of high background where the ethidium has not yet migrated out of the gel. They are however sufficient for many purposes, and do not generate as much ethidium waste (Your safety office will know what level of Ethidium causes a buffer to be declared a hazard).

Detecting Nucleic Acids with Nuclistain

Method II - Post Run Staining

1. Dilute Nuclistain stock 1:100 in distilled or deionized water. Prepare enough solution to completely submerge the gel. 2. Stain the gel with agitation for 20-30 minutes.

1. Prepare enough 0.5mg/ml EtBr in water or buffer to completely submerge the gel. This solution is stable for 1-2 months at room temperature in the dark.

3. Destain in deionized water. Bands will begin to appear within 15 minutes. Complete destaining for maximum sensitivity requires 3-12 hours.

2. After the run submerge the gel in the staining solution for 15-30 minutes (depending upon gel thickness). 3. Place gel on plastic wrap on a UV light box and observe under 300nm illumination. Bands will appear bright orange on a pale orange background.

Ethidium Bromide

The most commonly used stain for detecting DNA/RNA, ethidium bromide (EtBr) is a DNA interchelator, inserting itself into the spaces between the base pairs of the double helix. EtBr possesses UV absorbance maxima at 300 and 360 nm. Additionally, it can absorb energy from nucleotides excited by absorbance of 260 nm radiation. Ethidium re-emits this energy as yellow/orange light centered at 590 nm. The fluorescence of EtBr in aqueous solution is significantly lower than that of the intercalated dye. Ethidium bromide is a sensitive, easy stain for

Figure 4.1.1b Bands in gels stained with Ethidium Bromide fluoresce under ultraviolet light.

Notes: This protocol minimizes the amount of EtBr waste created with each gel run. Sensitivity the same as Method I can be achieved but may require destaining in water or 1mM MgSO4 to achieve the best sensitivity. In this method, bands become visible from the top and bottom of the gel as the dye diffuses into the matrix. High contrast results can often be achieved without destaining by soaking the gel until the top and bottom of the bands appear, and then leaving the gel to stand out of the staining solution for 15-30 minutes. During this time the stain will continue to diffuse into the gel, binding to the DNA at the expense of free dye. The result is a lower background without destaining. Always use plastic wrap under ethidium stained gels to avoid solarization damage to the surface of the transilluminator. USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Applications - Post Electrophoretic Analysis

Applications

Electrophoresis

Silver Staining

Silver staining is more sensitive than ethidium bromide for double stranded DNA, and detects single stranded DNA or RNA with no loss in sensitivity. Silver staining relies on the reduction of silver cations to insoluble silver metal by nucleic acids. This chemical reaction is insensitive to the macrostructure of the DNA molecule. Reduced silver grains deposit in the gel around the DNA bands, creating a “latent image.” The latent image is developed to visibility by soaking the gel in a solution of silver cations and a reducing agent. The silver granules in the latent image catalyze the further reduction and deposition of silver from the solution. Bands manifest as dark brown or black regions which appear before significant background develops. Development is stopped by altering the pH of the gel to a point where silver reduction is no longer favored. The mechanism whereby nucleic acids reduce silver is not well defined. Staining is enhanced by treatment with oxidants, and it may be that such treatment oxidizes vicinal sugar diols to highly reductive aldehydes, which are known to reduce silver (e.g. Tollens test). However, if this were the primary mechanism, silver staining would be expected to destroy the DNA. Recent reports indicate that enough DNA survives silver staining to provide a template for PCR amplification. This may reflect either the nondestructive nature of silver staining or the extreme sensitivity and power of the PCR reaction. Protocol 4.1.1c Silver Staining with the Sterling Silver Kit For mini-gels (10X7cm), use 100ml of each solution. For larger gels, increase STERLING volumes appropriately to immerse gel to depth of 1cm. Wash mini-gels in 200ml volumes of water, and agitate continuously during all steps. Glassware must be clean, and the water should be distilled or high-quality deionized. FIX 1. Incubate the gel for 25 minutes in 100ml of the standard mixture of 5:5:1 methanol:water:acetic acid. 2. Decant fixative, then add reconstituted STERLING Fixative (45ml water, 50ml methanol, 5ml STERLING Fixative Concentrate) and fix for an additional 5 minutes. WASH 1. Rinse the gel twice for 15 minutes in deionized water. The addition of 0.1% nonionic surfactant will aid in submerging the gel. 2. While the gel is washing, prepare Staining Solution (see below). Do not combine the two component solutions until just prior to use. PREPARATION OF STAINING SOLUTION

4.1.2 Blotting Nucleic Acids Northerns and Southerns

A DNA or RNA probe will selectively hybridize with nucleic acid molecules of complementary sequence in a sample. A labeled nucleic acid molecule of known sequence can facilitate detection of any complementary molecules in an unknown sample. This is the basis of the RNase protection assay (Section 2.1.8), and PCR amplification (Section 2.2.3), among other techniques. In theory, labeled nucleic acid molecules could act as specific “stains” for DNA or RNA species in gels. Only complementary fragments would be “stained”. However, specific hybridization requires nucleic acid polymers of twenty-five or more bases, which are too large to diffuse rapidly into a gel. In order to circumvent this problem, a method was devised to “print” an electrophoretic pattern onto a solid support, preserving the positional information from a gel, but removing the matrix. This process—blotting—was first publicized by Southern (1975). Southern demonstrated that DNA could be electrophoretically fractionated, transferred to nitrocellulose, and then probed with radioactively labeled DNA sequences, which would hybridize to their cognates on the membrane. The result, dubbed a “Southern Blot,” reveals a pattern of bands showing the size and relative amount of DNA containing the probe sequence. Soon thereafter RNA was blotted successfully (Northern blotting) and protein (Western blotting). The advantage gained by blotting was the immobilization of the electrophoretic pattern, rendering the molecules in that pattern accessible to macromolecular probes.

Transfer Techniques

Blots are created by laying a membrane over one face of the gel and then creating a flow which carries the molecules in the gel onto the membrane. The two most common methods used for Northern and Southern blotting are capillary flow and vacuum transfer. Electroblotting is also used occasionally, although it requires special care to prevent crushing or melting of the agarose gel. Capillary flow transfers are carried out in a dish of buffer (Figure 4.1.2a). The gel is placed on a porous support (usually filter paper or a sponge) that holds the gel above the buffer while allowing buffer to flow up to the gel. The membrane is placed on top of the gel, and a stack of absorbent paper is placed over the membrane. A weight is placed on top of the stack to ensure continued close contact of all components, and the entire assembly is left to stand for 12-16 hours. During this period, buffer is wicked out of the gel through the membrane and onto the dry filters above. As buffer flows into the gel from the tank below, the nucleic acids in the gel are carried upward to the membrane. Conditions are chosen to favor binding to the membrane, so molecules of nucleic acid are trapped there. Usually 80-90% of the molecules in the gel can be recovered on the membrane by this procedure.

1. Dilute 25ml Reagent A with 25ml of water.

Stack of towels

2. Dissolve 2.8 grams of Reagent B in 50ml of water. STIR UNTIL COMPLETELY DISSOLVED (approx. 5-10 minutes). 3. Immediately prior to use, pour (1) into (2) while stirring, and pour over gel. The combined solution has a useful life of 20-30 minutes.

Gel Support block

Wick

STAIN 1. Decant wash solution and immerse gel in combined staining solution. 2. Bands will begin to appear in 5-10 minutes. When desired intensity is achieved, stop development by immersing the gel in a 5% acetic acid solution.

Note: A new level of sensitivity in DNA staining is claimed for a number of fluorescent DNA and RNA dyes. These dyes are less mutagenic and more sensitive than EtBr, at times approaching or surpassing silver staining in sensitivity.

Membrane

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Buffer

Figure 4.1.2a Capillary blotting.

Vacuum blotting also uses a flow of buffer to elute bands onto the membrane, but it uses a vacuum instead of capillary action to create the flow (Figure 4.1.2b). The membrane is placed on a porous support, the gel is placed over it, vacuum is applied and the buffer is added to submerge the gel. This type of blotting is quite fast (1-3 hrs.), and gives excellent recoveries. Its only drawback is the need for specialized apparatus.

Electrophoresis Applications - Post Electrophoretic Analysis

Buffer Gel

Northern Blotting Membrane

Protocol 4.1.2a

ELECTROPHORETIC SEPARATION (see Protocol 2.4.6b) RNA samples should be free of contaminating particles and DNA, with an A260/A280 ratio of 1.9-2.0.

2. Visualize the gel on UV and photograph with a phosphorescent ruler.

Electroblotting of DNA or RNA from agarose gels is most often carried out in a “semi-dry” apparatus, between solid flat electrodes of metal or graphite. In this technique, potential is applied perpendicular to the gel, causing the nucleic acid fragments to eletroelute onto the membrane. With agarose, special care must be taken not to crush, distort or melt the gel during transfer. Electroblotting is far more popular for polyacrylamide gels in which capillary or vacuum techniques fail due to the smaller pore size. (Section 4.2.2 Western blotting). In the methods given for Northern and Southern blotting below, directions are given for setting up capillary blots. For instructions on vacuum or electro-blotting, see the protocols supplied by manufacturer’s of these devices.

Membranes

There are two popular membrane materials used in nucleic acid blotting. These materials are nitrocellulose (including supported nitrocellulose), and nylon (charged and uncharged). Nitrocellulose is the traditional support medium, and it is still employed by many researchers. In a high salt aqueous environment, nitrocellulose will bind to nucleic acids tightly but non-covalently. After transfer, nucleic acid binding is stabilized by baking the filter for 1 hour. The baked filter can be stripped and reprobed, but because the bound nucleic acid is not covalently linked to the membrane, significant signal loss can occur. A major drawback to nitrocellulose membranes is that they are quite fragile, requiring very careful handling to preserve the blot intact. Nylon filters have gained great popularity since their introduction, their most obvious advantage over nitrocellulose being their durability. Nylon filters can be handled roughly with no physical damage. The material withstands prolonged exposure to alkali, which allows use of the convenient alkaline transfer technique (Protocol 4.1.2c). Nylon is blocked by SDS, which simplifies the pre-hybridization steps. Nucleic acids can be covalently linked to nylon membranes through controlled exposure to UV light. The resulting blot may be stripped and reprobed many times with minimal loss of signal. Nylon membranes are available in uncharged form, or with immobilized positive charges on their surfaces. Positively charged nylon has a higher affinity for nucleic acids, which are anions, than does neutral nylon. Positively charged nylon will bind nucleic acid semi-permanently after alkaline blotting without UV cross-linking. However, in some cases, charged nylon may give higher backgrounds than uncharged membranes. Products for Northern and Southern Blotting AquaPor LE EC-202 AquaPor LE is a high quality, general purpose agarose ideal for most routine applications. (pg. 16)

SSC Buffer (20X) EC-873 Formulated with 18 MegOhm water. 0.2 micron filtration. More reproducible than bench-top buffers. (pg. 18)

SDS Solution 20% EC-874 For the same cost, avoids the discomfort and inconvenience of working with powdered SDS. (pg. 33)

ProgoGlow ECL CL-300 Highest sensitivity HRP chemiluminescent detection in Western and Southern blot techniques. (pg. 24)

Denaturation Solution EC-875 Neutralization Solution EC-876 Ready-to-use solutions for Southern blotting.

Formamide EC-608 Ready-to-use DNA denaturing agent. Redistilled, deionized, and stored under nitrogen.

(pg. 18)

Dextran Sulfate EC-877 Ultra pure reagent used in hybridization reactions to promote annealing and reaction times. (pg. 32)

(pg. 32)

TRANSFER 1. To remove formaldehyde and EtBr which may hamper transfer and/or recovery, soak the gel in 3 changes of DI water for 10 minutes each.

Applications

Figure 4.1.2b Vacuum blotting.

Electrophoresis

1. For highly expressed genes, load 10µg total of RNA. For trace expression detection, load 10µg of poly A+ RNA. Ethidium may be added to the gel (4 µl of 10 mg/ml per 100ml gel = 0.4µg/ml).

2. Place a platform slightly larger than the gel in the center of a 9X13X2” tray. Fill the tray with 10X SSC until the buffer is 0.5 cm below the surface of the platform (An inverted gel mold or a sponge make good platforms. Rinse commercial sponges before first use, to remove surfactants). 3. Cut a strip of Whatman 3MM paper to the width of the platform and length of the tray. Lay it over the platform so that the ends rest in the buffer. Roll a pipette over the filter to remove any bubbles beneath. 4. Flood the filter paper surface with 10X SSC and lay the gel on the filter paper. Roll the gel with a pipette if needed to remove air bubbles. 5. Place strips of plastic wrap on the platform around the gel to prevent buffer from bypassing the gel. Alternatively, lay a piece of plastic wrap over the gel and platform, and cut away the plastic over the gel with a razor blade, leaving a “mask” on the tray. 6. Flood gel surface with 10X SSC and carefully overlay membrane onto gel (Note: handle membrane with gloves or forceps and only at corners). 7. Cut 3 pieces of Whatman 3MM to the size of the gel. Wet one piece and lay it over the membrane. Roll the paper and membrane with a pipette to remove any bubbles. 8. Inspect carefully - gaps between gel and membrane block transfer and may create “hot spots” on the blot, making interpretation more difficult. 9. Wet each remaining cut piece of 3MM paper, lay over the stack, and roll to remove bubbles. 10. Lay a stack (3-4” high) of paper towels, cut to the size of the gel, over the top of the stack. 11. Place a glass plate and a 500g weight on top of the stack. Allow to transfer overnight. POST TRANSFER PROCESSING 1. Disassemble capillary stack down to membrane, mark well positions with indelible pen or pencil before removing membrane from gel. Note: Gel will be flattened to ~ 2mm thickness. The gel can be stained with EtBr and checked under UV to determine extent of transfer. 2. Rinse membrane in 5X SSC for 5 minutes at room temperature. 3. Cross-link RNA to membrane: Nylon - expose to UV (~150 mj/cm2). Nitrocellulose - Bake at 80°C for 120 minutes. 4. (Nylon filter only) Wash filter in 1X SSC + 0.1% SDS @ 65° C for 1 hour. This will substantially reduce the background. HYBRIDIZATION 1. Probe purification (Section 2.4.3) 2. Prehybridize in hybridization solution (see below) for 1 hour @ 65°C, then replace with fresh solution containing probe for hybridization. Many different protocols exist for hybridization reactions and each must be optimized for any given probe. General guidelines are given below.

Hybridization solution: 5X Denhardt’s Solution 100µg/ml Salmon or Herring Sperm DNA 0.1% SDS 5X SSPE 50% formamide Filter sterilize and store at -20°C. Denhardt’s solution (50X): 1% BSA 1% Polyvinylpyrrolidone 1% Ficoll SSPE (20X): 3M NaCl 0.2M Sodium Phosphate, pH 7.4 25mM EDTA 3. Select a hybridization temperature which will allow annealing of the probe, but prevent nonspecific binding to nontarget sequences. Note that high stringency washing later in the protocol may not be able to compensate for too low a hybridization temperature. The result will be a large number of false positive bands. continued

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Electrophoresis Applications - Post Electrophoretic Analysis

Applications

Electrophoresis

continued

POST TRANSFER

Calculation of theoretical melting temperature (Tm)

a. TmDNA: RNA = 79.8°C + 18.5 (log10[Na]) + 0.58 (%GC) + 11.8 (%GC)2 - 0.50 (%F) - (820/length) b. Tm decreases by ~ 1° C for every 1% increase in mismatches. c. Tm decreases by 0.5° C for every increase of 1% in formamide (%F). d. A good hybridization temperature to begin with is ~ 20°C below the calculated Tm. e. Washes should be carried out at ~15°C below the calculated Tm.

After hybridization, non-specifically bound probe is removed by washing in low salt buffer at high temperature. The salt concentration and temperature must be optimized for each probe/sample combination. A good starting point is to wash 1x 20 min in 1X SSC, 0.1% SDS at 45° C, followed by 3 x 20 min in 0.2X SSC, 0.1 % SDS at 65° C.

AUTORADIOGRAPHY See Section 4.1.2. STRIPPING

After autoradiography, the probe may be stripped from the blot, allowing the blot to be re-probed up to 5 times (nylon) or 2 - 3 times (Nitrocellulose). Incubate the blot in 50% formamide, 6X SSC at 65° for 30 - 60 minutes. Wrap stripped blot in plastic wrap and place on film overnight to confirm probe removal. Note: If blot is allowed to dry with probe bound to it, the probe will become permanently attached.

Southern Blotting

Protocol 4.1.2b

The protocols given are for Genomic Southern Blotting. This set of protocols will give superior results in any standard Southern application.

Genomic DNA samples must be extremely pure, A260/A280 = 1.8, with no nuclease contamination and minimal fragmentation.

RESTRICTION DIGESTION

Digests must be complete for results to be interpretable. It is advisable to run test reactions for the first blot, to optimize conditions and times. Incomplete digests are the primary cause of Southern Blot failure. It is particularly important to ensure that DNA has fully dissolved prior to digestion (allow 2 - 3 hours @ 4° C).

3. Fix the DNA to the filter: Nitrocellulose: Bake @ 80° C for 2 hours under vacuum, between layers of 3MM paper. Nylon: Cross-link DNA to the filter with UV irradiation (120mj/cm2).

Note: At this stage, filters may be stored dry in the dark for later probing.

1. Probe purification (Section 2.4.3) 2. Prehybridize in hybridization solution (see below) for 1 hour @ 65°C, then replace with fresh solution containing probe for hybridization. Many different protocols exist for hybridization reactions and each must be optimized for any given probe. General guidelines are given below.

Hybridization solution: 5X Denhardt’s Solution 100µg/ml Salmon or Herring Sperm DNA 0.1% SDS 5X SSPE 50% formamide Filter sterilize and store at -20°C Denhardt’s solution (50X): 1% BSA 1% Polyvinylpyrrolidone 1% Ficoll

SSPE (20X): 3M NaCl 0.2M Sodium Phosphate, pH 7.4 25mM EDTA 3. Select a hybridization temperature which will allow annealing of the probe, but prevent nonspecific binding to nontarget sequences. Note that high stringency washing later in the protocol may not be able to compensate for too low a hybridization temperature. The result will be a large number of false positive bands.

SAMPLE PREPARATION

2. Rinse filter in 6X SSC for 5 minutes to remove any agarose fragments.

HYBRIDIZATION

STRINGENCY WASHES

1. Disassemble the capillary stack. Mark the well positions on the membrane with pencil or indelible ink before removing it from the gel.

A 1 kb gene fragment is present in a typical mammalian genome at 0.3 ppm. This level of representation can be detected in ~ 10 µg of digested genomic DNA, using probes (length ~ 500 bp) labeled to 109counts/µg.

ELECTROPHORESIS (See Section 2.4.2 Restriction mapping) Include lanes of DNA markers, because digests of genomic DNA generate a smear, not discrete bands.

Calculation of theoretical melting temperature (Tm)

a. b. c. d. e.

TmDNA:DNA= 81.5°C +16.6 (log([Na]) + 0.41(%GC) - 0.63(%Formamide) - (600/length) Tm decreases by ~ 1° C for every 1% increase in mismatches. Tm decreases by 0.5° C for every increase of 1% in formamide. A good hybridization temperature to begin with is ~ 20°C below the calculated Tm. Washes should be carried out at ~15°C below the calculated Tm.

STRINGENCY WASHES

AUTORADIOGRAPHY

See Section 4.1.2. Southern Blots will need to be exposed between intensifying screens for up to 96 hours to achieve maximum sensitivity.

1. Use 0.7% of a low EEO Agarose, run in 1X TBE.

STRIPPING

2. Photograph gel after EtBr staining using a fluorescent ruler as a reference scale.

TRANSFER 1. Soak gel for 10 minutes in 5 volumes of 0.2N HCl. Soaking may be stopped when Bromophenol blue dye begins to turn yellow, indicating that the pH of the gel has dropped. Do not soak longer than 10 minutes.

After autoradiography, the probe may be stripped from the blot, allowing the blot to be re-probed up to 5 times (nylon) or 2 - 3 times (Nitrocellulose). Incubate the blot in 50% formamide, 6X SSC at 65°C for 30 - 60 minutes. Wrap stripped blot in plastic wrap and place on film overnight to confirm probe removal. Note: If blot is allowed to dry with probe bound to it, the probe will become permanently attached.

2. Rinse gel in 5 volumes of deionized water for 5 minutes.

Note: This step depurinates scattered sites on the DNA. These sites are cleaved by the alkaline treatment below, enhancing the transfer of fragments >10kb. If all fragments of interest are known to be < 10kb, this step may omitted.

3. Soak gel for 45 minutes in denaturation solution (1.5M NaCl, 0.5M NaOH) at room temperature with gentle agitation. 4. Rinse in deionized water 1 - 2 minutes. 5. Soak gel for 30 minutes in neutralization solution (1M Tris pH 7.4, 1.5M NaCl) with gentle agitation. 6. Set up capillary stack as for Northern Blotting (Protocol.4.1.2a) using 10X SSC buffer. For recovery of small fragments < 500 bp, use 20X SSC. Note that nylon will bind small fragments better than Nitrocellulose. 7. Allow to transfer overnight. continued

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Alkaline Blotting

Protocol 4.1.2c

Positively charged nylon membranes allow the use of alkaline transfer buffers, which link the nucleic acids to the membrane without UV crosslinking. In some cases, alkaline blotting gives a higher background. Increasing the concentration of blocking reagent will often eliminate this problem.

NORTHERN BLOTTING

Follow the basic procedure given in protocol 4.1.2a using 40mM NaOH in place of the 10X SSC.

SOUTHERN BLOTTING

Follow the procedure in protocol 4.1.2b, omitting step 5, and using 0.4M NaOH in place of 10X SSC.

Electrophoresis Applications - Post Electrophoretic Analysis

4.1.3 Autoradiography

Autoradiography is the use of X-ray (or occasionally photographic) film to detect radioactive materials. It produces a permanent record of the positions and relative intensities of radiolabeled bands in a gel or blot. Typically, biomolecules are labeled with 32P or 35S and detected by overnight film exposure.

Autoradiography Detection Limits Isotope

Minimum CPM for Detection 7

Energy per Emission (MEV)

>10

0.0055

2000

0.050

1000

0.167

100

0.70

100

(gamma)

125

Table 4.1.3a Figure 4.1.3a Typical autoradiographic technique involves enclosing the gel or blot with X-ray film for at least 24 hours within an X-ray cassette. Development of the film reveals the gel or blot image. Note the use of radioactive or phosphorescent dots to record the orientation of the film. Table 4.1.3a gives the amounts of commonly used isotopes which can be detected by overnight autoradiography.

Because water quenches beta particle emissions, optimum sensitivity is obtained using dried blots and gels . However, because drying blots permanently fixes the probe onto the membrane, if blots are to be stripped and re-probed, DO NOT DRY. Wrap in plastic wrap to prevent water and radioactivity from contacting the film. Note that 35S and 14C emit β-particles at low energy, and the majority of such particles are unable to penetrate a layer of plastic wrap. For 35S or 14C detection place the dry gel or blot directly on the film or use fluorographic enhancement. Use high quality X-ray film: Kodak XAR or equivalent. 1. Prepare gel or blot. 2. Wrap wet samples in plastic wrap. 3. Tape the sample to the inside face of an X-ray cassette, with enhancement screens for 32P if desired. It is essential to fasten the sample so it does not move, because it must later be aligned with the exposed film. 4. Fasten a phosphorescent ruler or other orientation aid (radioactive dots) along one side. If a marker other than a ruler is used, be sure to include enough marks to unambiguously align the developed film to the sample. 5. In the dark, or under safelight, place one piece of X-ray film over the sample. For 35S or 14C, room temperature exposure is sufficient. For 32P, intensifying screens require a temperature of -70° C for exposure. 6. Expose film. Use Tables 4.1.3a & b as a guide for length of exposure. A good rule of thumb is that bands must be detectable on a Geiger counter for overnight exposure to be sufficient. 7. To avoid condensation on the film allow the cassette to return to room temperature before opening. Develop the film according to manufacturer’s instructions.

Radioactive Exposure of Film

β-particles emitted by radionuclides penetrate film emulsions to a depth proportional to their energy. As these particles pass through the film, they activate the silver halide crystals in the emulsion. Activated crystals are detected by reduction to black silver grains in the development step. The activated silver halide crystals are not stable, although they can be stabilized by further exposure to β-particles, or to light. On average, a crystal will require 5 “hits” from either radioactive emissions or light to be stably activated, and thus detected. In order to compensate for this instability, autoradiography is often carried out at –70°C, which stabilizes the activated crystals, enhancing sensitivity. Additional sensitivity is gained by “preflashing” the film before use. The film is exposed to a microsecond burst of light which is calibrated to bring the crystals in the film to a partially stabilized, activated state. Preflashed film requires only one “hit” per grain deposited. This not only increases sensitivity but also ensures that the film signal will be linear with the amount of radioactivity, even at very low signal levels.

Fluorography

Sensitivity of autoradiography can be greatly enhanced through the use of fluorography, which converts radioactive emissions into light. Light penetrates film more efficiently than β-particles and is therefore more readily detected. Various phosphor compounds are available which can be dried into a gel, and which absorb the energy from β-particles and re-emit it as light. For high energy β- particles, though, such as those from 32P, the problem is not insufficient penetration but over-penetration, in which the emitted particles pass entirely through the film without activating the emulsion. To recover the energy of these “lost” particles, phosphorescent intensifying screens are used, which emit light on irradiation. Phosphorescent intensifying screens require low temperatures (-70°C) for optimum performance.

DPM/ band

Beq./ band

Exposure (hours)

500

8.3

48-72 24

Gel % 3

5000

C/35S

300

C/35S

1000

8-12

Autoradiographic Enhancement with Autofluor National Diagnostics’ Autofluor is an extremely sensitive, water based fluorographic enhancer for autoradiography on gels, TLC plates or paper chromatograms.

GELS 1. After staining, fix the gel with 5% glacial acetic acid, 5% isopropyl alcohol, and 90% water. Fix for 15 to 20 minutes. Pour off fixing solution and discard according to radioactive disposal procedures. 2. Rinse the gel in a continuous flow of water for 15 minutes to ensure the complete removal of any acetic acid residue. To prevent crystal formation, it is important that the gel be thoroughly rinsed after fixing. Should the gel develop white crystals on contact with Autofluor, dissolve the precipitate by soaking the gel in a solution of 1g sodium carbonate/100ml water or in Tris Buffer. Soak the gel in Autofluor until the white precipitate is fully dissolved. Repeat from the beginning of step 2.

3. Cover gel with Autofluor until the depth of Autofluor is twice the thickness of the gel. Gently agitate in Autofluor for 30 min/mm of gel thickness. Pour off remaining Autofluor and retain for future use. Label reserved material as radioactive. Autofluor may be reused several times before a diminishing response is observed. 4. DO NOT WASH GEL. Place directly on filter paper and dry on gel dryer under heat (80oC) and vacuum. 5. The gel will have a white to light tan sparkling appearance similar to freshly fallen snow. 6. Place on film and expose at -70oC. Due to the higher light output of the Autofluor phosphor, less exposure time is needed for gels treated with Autofluor than for gels treated with PPO/DMSO. Sufficient exposure time for a 5000 dpm/band is 24 hours. Overexposure of the film will cause the bands to become fuzzy and resolution to be lost. 7. Develop film according to manufacturer’s instructions. PAPER CHROMATOGRAPHY AND TLC PLATES 1. Spray twice or dip plates in Autofluor and allow to dry. 2. Place on film and expose at -70oC.

Fluorography Exposure Times Isotope

Applications

Gel or blot

Protocol 4.1.3a

Electrophoresis

X-ray film

Autoradiography

TO MAXIMIZE AUTOFLUOR EFFICIENCY

Autoradiography Enhancement Autofluor LS-315 National Diagnostics’ autoradiographic image intensifier, Autofluor, is a water based phosphor that yields superior results to PPO-DMSO. (pg. 30)

• If gels crack or stick during drying, add 0.5% (5ml/liter) of glycerol directly to the Autofluor before using. • Since Autofluor is inducted into the gel by crystallization in situ as opposed to precipitated, it is advantageous to form the smallest crystals possible. This is accomplished by drying as quickly as possible under the strongest vacuum possible. A vacuum pump with a good seal on the dryer is preferred over a “house vacuum.” After the gel appears dry, turn off heat and continue vacuum for another 1/2 hour.

Table 4.1.3b USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Applications - Post Electrophoretic Analysis

Applications

Electrophoresis

4.2 Post-Electrophoretic Protein Detection The techniques for visualizing proteins following electrophoresis may be divided into three broad categories: Immunological detection, activity stains, and chemical staining methods (discussed below). In turn, chemical staining breaks down into three subgroups: Visible stains, fluorescent stains, and metal-deposition stains (e.g. silver stains). Most chemical stains require a fixation step prior to, or combined with the actual staining process.

4.2.1 Fixing Proteins on Gels

Fixing (or fixation) is the process whereby proteins are denatured and precipitated in large insoluble aggregates within the gel matrix. Fixation accomplishes several goals. Primarily, fixation prevents the diffusion of proteins, thus keeping the protein bands sharp and resolved during the staining process. In addition, fixation removes gel buffer components, most importantly SDS, which may interfere in the staining process. In some cases, fixatives are used which modify the proteins to enhance the staining reaction. An ideal fixative is fast, convenient and nonhazardous to use, and preserves the fine detail of the gel. It is important to be aware that fixing a protein within a gel drastically lowers the amount of protein which can be recovered from that gel after bands have been identified (see guide strip technique, Section 4.2.2). This is probably due to the trapping of gel matrix strands within the denatured protein complexes. All fixatives operate by causing precipitation of the protein by converting it to an insoluble form. The most commonly used fixatives are solutions of short chain alcohols and acetic acid in water. The combination of low pH and high organic solvent content disrupts the hydrogen bonding which holds protein structures together, and exposes hydrophobic portions of the protein core. The result is an uncoiling of the peptide chain, followed by an essentially irreversible association between chains, producing a high molecular weight complex which is trapped inside the gel. This family of fixatives is cheap and relatively nonhazardous (depending on the alcohol used), and has the additional advantage that many stains are soluble in the fixative. This allows the combination of fixing and staining in one step. The only major drawback is that these solutions are only moderately denaturing, and may not fully fix small or unusually soluble proteins.

Native proteins

SDS denatured

Insoluble monomer

Figure 4.2.1a Fixing proteins with acetic acid and alcohol results in an uncoiling of the peptide chains to produce insoluble complexes and monomers.

Polymeric mass

Stronger fixatives include trichloroacetic acid (12% in water), sulfosalicylic acid, or formaldehyde. TCA, sulfosalicylic acid and other strong acids act by protonating weak acids in the protein structure, disrupting the salt bridges and charge interactions required to maintain protein secondary structure. Aldehydes, such as formaldehyde and glutaraldehyde, react with amines on the surface of proteins, creating covalent cross links between protein molecules, resulting in a truly irreversible denaturation.

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Protocol 4.2.1a Fixing Proteins on Electrophoresis Gels Most protein gels can be fixed effectively by soaking for 1 hour in 45% methanol, 45% water, and 10% glacial acetic acid. This solution is stable for 30 days at room temperature. A more stable fixative is 25% isopropanol, 65% water and 10% acetic acid, which can be stored at 4Â°C for up to 4 months. NOTES: In all cases, agitation during fixing will speed the process by encouraging the penetration of the gel by the fixative. As fixing elutes SDS and other interfering components from the gel, sufficient fixative should be used to dilute these components by at least 5:1. Use fixative only once.

Fixing Difficult Proteins

Small or unusually soluble proteins may not be sufficiently fixed by the above protocol. As these proteins diffuse through and out of the gel, smeared bands and loss of sensitivity may result. Prefixing of the gel in 12% trichloroacetic acid for 1-3 hours at room temperature prior to fixing by the above protocol will generally improve the fixing, and hence the staining of such proteins. In certain cases, where proteins are heavily glycosylated or strongly basic, acid based fixatives may be ineffective. Small peptides may also be resistant even to strong acid fixatives. In such cases an effective alternative to acid precipitation is covalent cross-linking of the proteins with formaldehyde or glutaraldehyde. Formaldehyde fixation may be accomplished in a solution of 25% Ethanol, 15% Formalin (Formalin is 35% formaldehyde), 60% water. Gels are submerged in this solution for 1 hour, and may then be stained with or without subsequent alcohol/ acetic acid fixation. Glutaraldehyde is generally used as a fixative in Silver Staining. Gels are soaked in 10% aqueous glutaraldehyde for 30 minutes, then washed for 2 x 20 minutes with water before staining. This denatures the proteins and fixes them in the gel; it also puts reactive aldehyde groups on the surface of the proteins, which enhance the silver stain reaction.

4.2.2 Staining Proteins in Gels

Chemical stains detect proteins based on differential binding of the stain by the protein molecules and the gel matrix. They are nonspecific in action, detecting proteins without regard to their individual identities. The important characteristics for a useful stain are low background, high sensitivity, large linear range and ease of use. A variety of stains are available which offer good performance in all of these areas. Most stains in use today offer essentially zero background after destaining steps of various lengths and complexities. This gives a high signal to noise ratio for the low end of the detection range, enhancing sensitivity and linearity. The overall sensitivity of a given dye depends upon its extinction coefficient and the avidity with which it binds to protein. Coomassie Blue R-250, the most commonly used protein stain, can detect as little as 0.1 Âľg of protein per band on a gel. Silver staining can detect down to 1 ng of protein, or less in some cases. In evaluating stains, remember that a particular stain will not detect all proteins equally well. Coomassie Blue will detect 50 ng of some proteins, while other proteins require 10 times this amount to give a discernible signal. This often leads to errors of interpretation in reading stained gels: the (mistaken) assumption that all proteins are detected with equal sensitivity leads to over- or underestimation of protein levels. The sensitivity with which a dye detects a given protein in turn determines the linear range of detection for that protein. Within the linear range, the relative intensity of a protein band relates directly to the relative amount of protein in that band. In general, proteins lose linearity as the amount of protein in a band increases. The color immobilized on that band saturates, and further increases in dye binding do not lead to discernible increases in color intensity. Proteins which are detected with greater sensitivity will saturate first, while those which do not give a perceptible signal at low concentrations will be linear in the higher concentration ranges.

Electrophoresis Applications - Post Electrophoretic Analysis Ease of use varies considerably from one stain to another. Coomassie staining, for example, requires up to 16 hours to complete, but requires minimal “hands on” time. In contrast, silver staining can be completed in one hour, but requires intense worker involvement. There are variations on most staining techniques which trade time or convenience for sensitivity, or sensitivity for time.

Time Required

Amido Black

Xylene Cyanole Sensitivity (nucleotides)

500 ng

12 hrs

STANDARD PROTOCOL - COOMASSIE BLUE R-250 1. Gel may be prefixed in 50% MeOH, 10% HOAc, 40% H2O for 30 minutes to overnight. 2. Stain gel in the above solution, with 0.25% Coomassie Blue R-250, for 2 - 4 hours, until the gel is a uniform blue color. Staining is complete when the gel is no longer visible in the dye solution. Prior to complete staining, the gel will appear as a lighter area against the dark staining solution. 3. Destain for 4 - 24 hours in 5% MeOH, 7.5% HOAc, 87.5% H2O. Bands will begin to appear in 1 - 2 hours. Destain until background is clear.

This method will detect as little as 50ng/band.

Coomassie Blue R-250

16 hrs

50 ng

4. Store gels in 7% HOAc.

Coomassie Blue G-250

1-2 hrs

200 ng

RAPID PROTOCOL - COOMASSIE BLUE R-250

Colloidal Coomassie

1-8 hrs

5 ng

Immunological

4-6 hrs

<10 pg

Insite System

30 min

10 ng

2 hrs

1 ng

10 min

10 ng

Silver Zinc

Applications

Gel % Stain

Staining Gels with Coomassie Blue R-250 or Coomassie Blue G-250

Electrophoresis

Protein Staining Techniques

Protocol 4.2.2a

1. Fix gel in 25% IPA, 10% HOAc in water, 30 - 60 minutes. 2. Stain gel in 10% Acetic Acid in water, containing 60 mg/L of Coomassie Blue R-250. Bands will appear in 30 minutes. Allow staining to proceed until desired band intensity is reached. In this protocol, background staining is low due to the very low dye concentration used. 3. Destain gel in 10% Acetic Acid for 2 hours or more. Store gels in 7% HOAC. RAPID PROTOCOL - COOMASSIE BLUE G-250

Table 4.2.2a

Mechanism of Staining

With the exception of silver staining, chemical stains operate by binding to proteins with a higher affinity than to the gel matrix. The result is a local increase in concentration of the dye in the protein bands. Chemical staining is usually a two step procedure. The gel is first saturated with the dye. Then, excess dye is washed out under conditions which favor dye binding to protein. The three most commonly used dyes for in-gel staining are Amido Black, Coomassie Blue G-250, and Coomassie Blue R-250.

Coomassie Blue

The Coomassie dyes (R-250 and G-250) bind to proteins through ionic interactions between dye sulfonic acid groups and positive protein amine groups as well as through Van der Waals attractions. Coomassie R-250, the more commonly used of the two, can detect as little as 50 ng of protein. Though less sensitive, Coomassie G-250 can be used in place of the R-250 form to create a rapid and convenient staining procedure. This capability of G-250 is due to its particular properties. Coomassie G-250 manifests a leuco form below pH 2. Solutions of the dye, dark blue black at pH 7, turn a clear tan upon acidification. The leuco form recovers its blue color upon binding to protein, apparently due to the more neutral pH of the environment around the protein molecule. Under proper conditions, a gel placed in an acidified solution of Coomassie G-250 will manifest blue protein bands on a light amber background. The bands develop rapidly and there is no need to destain, for the background color is so light as to be essentially clear. This stain is less sensitive than Coomassie Blue R-250 protocols, detecting 200 ng of most proteins. The loss in sensitivity is offset by the speed and convenience of the protocol, which saves up to 11 hours versus the most sensitive R-250 procedures.

1. To make the Coomassie Blue G-250 staining reagent, dissolve 0.2g dye in 100 ml H2O (this will require warming to ~ 50°C). Cool and add 100 ml 2N H2S04. Incubate at room temperature 3 hours to overnight, then filter. To filtered solution, CAREFULLY add 22.2 ml 10N KOH, then add 28.7g TCA. Allow to stand > 3 hours, then filter again if necessary to obtain an amber-brown solution without blue precipitate. 2. To stain, immerse gel in above solution. Bands will begin to appear within 15 minutes. Intensity and sensitivity will continue to improve for several hours. 3. Staining solution is stable for 2 - 3 weeks @ 25°C.

Colloidal Coomassie

The capacity of Coomassie Blue G-250 to form metastable colloidal suspensions has been exploited to create a stain which is substantially more sensitive than the standard Coomassie R-250 protocol. In high salt, acidic solutions, the G-250 dye disperses to form a uniform colloid. The particles of this colloid have two useful properties. First, they are too large to enter into most gel pores, so submerging a gel in the dye suspension does not create a high background color. Second, the particles break down upon contact with proteins, releasing soluble dye. When a gel is submerged in a colloidal suspension of Coomassie Blue G-250, particles of dye essentially dissolve into the protein bands, resulting in an accumulation of color only where protein bands are present. Destaining is not required. Colloidal Coomassie stains are capable of detecting as little as 5ng of protein per band. Although the development of bands below 20 ng may take up to 8 hours, the “hands on” time of this type of stain is generally less than 10 minutes. Because of its ease and sensitivity, colloidal Coomassie staining has gained a great deal of popularity.

Products for Staining Proteins in Gels ProtoStain Blue EC-727 Easy-to-Use, Eco-Safe Colloidal Coomassie Stain. Detects as little as 1ng of protein per band. (pg. 21) Coomassie Blue R-250

Coomassie Blue G-250

Figure 4.2.2b The two Coomassie Blue dyes, R-250 and G-250, differ by two methyl groups.

Coomassie Blue G-250 HS-605 Converted to a leuco form at low pH, used in colloidal staining techniques. (pg. 23)

STERLING Rapid Silver Stain EC-720 The fastest and most sensitive silver stain to date, producing superb results in one hour. (pg. 22)

Coomassie Blue R-250 HS-604 Coomassie Blue R-250 is the time-tested standard for protein visualization. (pg. 23)

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Electrophoresis Applications - Post Electrophoretic Analysis

Protocol 4.2.2b

Applications

Electrophoresis

Procedures for Using ProtoStain Blue ProtoStain Blue stain formulation is compatible with all polyacrylamide gel types, and produces lower background staining than competing stains without requiring extensive water destaining to produce a crystal-clear background. ProtoStain Blue is ready to use- no measuring or mixing required. STANDARD PROTOCOL As llittle as 1ng of denatured BSA can be detected by this protocol. 1. Wash the gel 3 times for 10 minutes each with deionized water on an orbital shaker. Decant wash solution. 2. After the last wash, add enough ProtoStain Blue solution to completely cover the gel. 3. Bands containing more than 1µg of protein will be detected within 15 minutes. For full sensitivity incubate the gel in the stain for at least 4-5 hours. Longer incubations in the stain will not adversely affect the gel or the staining sensitivity. 4. Remove the stain and wash the gel in deionized water. Incubating the gel in water increases the sensitivity of detection by reducing the background to crystal clear. The gel is stable in water for up to a week without loss of sensitivity. There is no need to store the gel in a salt solution.

RAPID PROTOCOL (60 minutes) For fast staining - complete in 60 minutes. 20ng of denatured BSA can be detected after 10 minutes destaining in water. Less than 5ng can be detected after overnight incubation in water, due to a combination of bands binding residual dye and the production of a crystal clear background. All steps are performed in a loosely covered plastic container. This protocol is optimized for 0.75mm thick laemmli formulation mini gels. 1. Wash the gel by heating to 95°C in deionized water for 45 seconds to one minute. Incubate for an additional minute on an orbital shaker. 2. Repeat the above step two more times. After the last wash rinse the gel in cold deionized water. Decant rinse water. 3. Warm enough ProtoStain Blue solution to 65°C to completely cover the gel. Add warmed stain to the gel. 4. Shake the gel in the stain on an orbital shaker for 10-50 minutes. Gels thicker than 0.75mm may require longer incubations. Remove the stain and rinse the gel several times. Incubate the gel in water on an orbital shaker until the required contrast/sensitivity is achieved.

PREPARING SAMPLES FOR MASS SPECTROSCOPY Gels stained with ProtoStain Blue can be destained for in-gel tryptic digests with 25-100mM ammonium bicarbonate/50% acetonitrile. Procedure: 1. Cut out gel band or spot. Cut band into 1mm x 1mm pieces if necessary. Place in an eppendorf tube. 2. Add 200μl destaining solution to gel pieces. 3. Incubate at room temperature or 37°C for 30-45 minutes. 4. Remove destaining solution. 5. Repeat steps 2-3. Gel pieces should now be transparent.

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Fluorescent Stains

Various stains have been developed over the past decade which offer fluorescent detection of proteins in gels. These compounds show an increase in fluorescence upon protein binding. The sensitivity of these stains varies from the range of standard Coomassie staining to better than Silver staining. The protocols for fluorescent stainng are generally rapid and straightforward, but acceptance of these stains has been limited by the need for a UV light box and photographic system, and the lack of permanent staining on the gel.

Zinc Stains

Proteins contain many chemical groups which can bind metal ions. A number of stains have been developed which take advantage of this capacity. Zinc staining is the most sensitive and convenient representative of this group of detection systems. In zinc staining, the gel is first impregnated with imidazole, and then placed in a solution of a zinc salt. The bivalent zinc forms an insoluble complex with the bidentate imidazole. Precipitation of this complex turns the gel an opaque white. Protein bands sequester the zinc in their vicinity, preventing formation of the precipitate. The result is clear bands on an opaque white gel, with sensitivities down to nanogram levels. One significant advantage of this technique is that it is non-denaturing and completely reversible. It is thus ideal for applications where recovery of intact, unaltered protein is desired.

Electrophoresis Applications - Post Electrophoretic Analysis

Silver Stains

Utilizing the same chemistry as black and white photography, silver staining is another highly sensitive method for the visualization of protein bands on electrophoresis gels. Silver ions are reduced to insoluble silver metal granules in the vicinity of the protein molecules. Sufficient silver deposition is visible as a dark brown or black band on the gel.

Protocol 4.2.2e Silver Staining with the Sterling Silver Kit For mini-gels (10x7cm), use 100ml of each solution. For larger gels, increase STERLING volumes appropriately to immerse gel to depth of 1cm. Wash mini-gels in 200ml volumes of water, and agitate continuously during all steps. Glassware must be clean, and the water should be distilled or high-quality deionized.

FIX 1. Incubate the gel for 25 minutes in 100ml of the standard mixture of 5:5:1 Methanol:Water:Acetic Acid. 2. Decant fixative, then add reconstituted STERLING Fixative (45ml water, 50ml methanol, 5ml STERLING Fixative Concentrate) and fix for an additional 5 minutes.

Applications

The initial deposition of silver is referred to as a latent image. This “image” is not visible, as the number and size of the silver granules deposited at this stage are minute. In the next stage a moderate reducing environment is created in the gel, in which all of the silver ions begin to “plate out” slowly. The silver granules which make up the latent image act to catalyze this process, resulting in more rapid deposition of silver at the sites of the protein bands. Silver staining is thus a kinetic process: development must be monitored and stopped at the point where the highest contrast between band and background is achieved.

In certain instances, the effects of staining a protein may interfere with subsequent analysis. Examples are Coomassie staining when enzymatic activity is required, or silver staining prior to amino acid analysis, when covalent modification of the amino acids will give spurious results. In these cases, it is common to use a “guide strip”. A guide strip is a lane which is run parallel to the lane to be analyzed, and containing either size markers or a duplicate sample. After the gel is run, the guide strip is cut off and stained, and then realigned with the gel and used as a template to guide band excision. The technique is straightforward. The only common error is to fail to re-equilibrate the gel with running buffer after staining. As many stains cause shrinkage or swelling of the gel, re-equilibration is necessary for accurate and consistent realignment.

Electrophoresis

The exact mechanism of silver staining is subject to debate, but certain key points are generally acknowledged. Staining appears to be dependent upon the initial reduction and resulting immobilization of a small number of silver ions by the proteins in the gel. This reduction may be caused by aldehydes created during fixing with glutaraldehyde or with oxidizing agents (e.g. chromic acid). Reduction of silver may also be enhanced by sequestration of the silver cations by carboxylic acid side chains, or by amino side chains, creating an amine-silver complex.

Guide Strip Technique

Figure 4.2.2b In the Guide Strip Technique, a parallel lane is excised and stained to guide band excision.

Staining of Proteins Immobilized on Membranes Immunological detection of proteins requires that proteins be transferred and immobilized onto a membrane support after electrophoresis (Section 4.2.3 Western Blotting). Staining of the immobilized proteins establishes transfer efficiency, and allows the operator to mark the membrane with the locations of lanes and size markers, facilitating later analysis. The mechanism of staining is the same as for in-gel staining. Conditions must be established under which the dye binds more avidly to the protein than to the support, resulting in dark, high contrast zones corresponding to the presence of protein. However, the requirements for the staining are different. Sensitivity is less of an issue, as markers are generally loaded in high concentration, and lanes of sample will show up even when individual bands in the lanes may be faint. Speed and protein recovery (not stripping the protein from the membrane) are more important in this case.

WASH

Protocol 4.2.2f

1. Rinse the gel twice for 15 minutes in deionized water. The addition of 0.1% nonionic surfactant will aid in submerging the gel. 2. While the gel is washing, prepare Staining Solution (see below). Do not combine the two component solutions until just prior to use. PREPARATION OF STAINING SOLUTION 1. Dilute 25ml Reagent A with 25ml of water. 2. Dissolve 2.8 grams of Reagent B in 50ml of water. STIR UNTIL COMPLETELY DISSOLVED (approx. 5-10 minutes). 3. Immediately prior to use, quickly pour (1) into (2) while stirring, and pour over gel. The combined solution has a useful life of 20 minutes. STAIN 1. Decant wash solution and immerse gel in combined staining solution. 2. Bands will begin to appear in 5-10 minutes. When desired intensity is achieved, stop development by immersing the gel in a 5% acetic acid solution.

Staining of Proteins Immobilized on Membranes PONCEAU S 1. To make a stock solution of Ponceau S, dissolve 0.5g Ponceau S in 100 ml of 1% Aqueous Acetic Acid. 2. Immerse blot in Ponceau S stock solution for 5 minutes. 3. Transfer blot to deionized H2O, and agitate until bands appear (1-5 minutes). 4. Mark bands with indelible ink (note: bands will fade in 15 minutes). INDIA INK 1. Wash blot in 0.3% Tween 20 in Phosphate Buffered Saline (PBS), 5 x 30 minutes @ 37°C. 2. Transfer blot to India Ink solution (0.1% Pelikan 17 Black or equivalent in 0.3% Tween 20 PBS) 3. Rinse membrane 2 times in Tween solution. Destain in Tween 20 solution until desired contrast is achieved. This protocol will detect as little as 50 µg per band. USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis Applications - Post Electrophoretic Analysis

Applications

Electrophoresis

4.2.3 Immunological Detection of Proteins

The highly specific binding interaction between antibodies and their unique antigens has been exploited to create sensitive and specific detection systems for proteins. An antibody can be raised and/or purified against a single epitope on an antigen. When antibody and antigen are mixed, the antibody will bind tightly to the epitope it recognizes. This identifies the antigen as bearing the epitope in question. Thus, immunological detection answers questions about the structure and identity of a protein which cannot be addressed through conventional chemical stains. In addition, immunological detection can be as much as 100 fold more sensitive than chemical stains.

Mechanism of Immunostaining

Antibodies, or immunoglobulins, are produced by the body in response to invasion by foreign proteins. The function of an antibody is to bind to a specific site (epitope) on the invader, thus tagging it for destruction by other agents of the immune system. The salient characteristics of antibody binding are binding strength and selectivity. In assay terminology, antibodies are said to be accurate (low false negatives) and specific (low false positives). In the body, the mere presence of a bound antibody on the surface of an antigen is sufficient signal for the immune system to “detect” that molecule. In an in-vitro assay, additional aids are required. These aids take the form of easily detectable molecules, which are bound to the antibody. Examples are enzymes, such as horseradish peroxidase (HRP) or fluorescent dyes. Enzymes are particularly suited to this role, because they are proteins, and thus stable wherever antibodies are stable. The catalytic cycling of enzymes greatly multiplies the sensitivity of the assay. The two enzymes most commonly used for labeling antibodies are horseradish peroxidase and alkaline phosphatase (AP). Chromogenic and luminogenic substrates are available for both of these enzymes.

Alkaline phosphatase

Chromogenic and luminometric substances are also available for HRP. The “classic” chromogen used with HRP is diaminobenzidine (DAB). In the presence of H2O2, HRP will oxidize DAB, creating a water insoluble brown precipitate. DAB has two primary disadvantages, it is relatively insensitive and it is a potential carcinogen which must be decontaminated before disposal. The sensitivity and convenience of HRP based detection has been greatly enhanced by the use of luminol as a chemiluminescent substrate. HRP catalyzes the reduction of H2O2 to H2O, and then recovers the electrons used for this reduction from luminol, generating an oxidized luminol radical. The luminol radical reacts with O2 to create O2–. The superoxide anion then reacts with a second luminol radical to produce a luminol endoperoxide, which subsequently decays with emission of light. The chemiluminescent reaction of HRP with luminol can be enhanced up to 104 fold by the inclusion of a variety of compounds, including substituted phenols, amines and napthalene derivatives. The mechanism of enhancement is still unclear, but it is theorized that the enhancers act as “electron conduits,” channeling electrons more rapidly along the series of reactions from HRP + luminol to light emission.

Figure 4.2.3a The basic method of immunostaining is to probe with an antibody which is bound to a detectable molecule such as an enzyme or a fluorescent dye.

Alkaline phosphatase catalyzes the removal of a phosphate group from its substrate. A variety of synthetic substrates have been constructed which, on phosphate hydrolysis, liberate chromogens or luminescent compounds. A commonly used chromogenic substrate is bromochloroindoyl phosphate (BCIP) in conjunction with nitro blue tetrazolium (NBT). Dephosphorylation of BCIP generates one half of an indigo dye molecule. Dimerization generates the indigo dye, and liberates two reducing equivalents which can reduce NBT to the blue insoluble formazan. This system generates a deep blue insoluble dye, which is ideally suited for blotting procedures. Alternatively, PNPP (para nitrophenyl phosphate) is hydrolyzed to liberate a soluble yellow dye, useful for ELISA reactions. Luminometric substrates for alkaline phosphatase are exemplified by CSPD (chloro-phospho-phenyl dioxetane). Upon dephosphorylation, this material yields a metastable charged dioxetane which then cleaves, releasing a highly energetic chlorophenylate. This molecule decays within 1 minute to a ground state, releasing light of wavelength 466 nm. 90

Horseradish Peroxidase

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Dephosphorylation

Figure 4.2.3c Mechanism of light emission by luminol, a luminescent substrate for use with alkaline phosphatase.

Products for Western Blotting

Cleavage

ProtoGlow ECL CL-300 Chemiluminescent system enhances horseradish peroxide visualization. Ideal for Western blotting. (pg. 24)

Light Emission

Figure 4.2.3b Mechanism of light emission by CSPD, a luminescent substrate for use with alkaline phosphatase.

Tris-Glycine Electroblotting Buffer EC-880 Stable, economical, ultra-pure. Absolutely reproducible performance. (pg. 19) ProtoGel EC-890 30% solution of Acrylamide and BisAcrylamide, 37.5:1 ratio. Filtered, Deionized, and Stabilized.

(pg. 8)

ProtoBlock System CL-252 To enhance signal to noise ratio during chemiluminescent membrane bound visualization techniques. (pg. 26) Buffers: Formulated with 18 MegOhm water. 0.2 micron filtration. (pg. 19) Tris-Glycine-SDS (10X) ProtoGel Resolving Buffer (4X) ProtoGel Stacking Buffer (4X) TBS (10X) TBST (10X) PBS (10X)

EC-870 EC-892 EC-893 EC-881 EC-882 CL-253

Electrophoresis Applications - Post Electrophoretic Analysis

Enzyme Linked Immunosorbent Assay (ELISA)

Antibodies are large molecules and penetrate gels slowly. In order to efficiently use immunological staining for post-electrophoretic detection, the proteins in the gel must be immobilized on an exposed surface, a process called “blotting.” Electroblotting is commonly used in the Western system. Following the run, the gel is sandwiched with a membrane and a voltage is applied perpendicular to the plane of the gel, such that proteins are electrophoresed out of the gel and onto the membrane.

Applications

Various membranes are employed in Western blotting, but the two predominant types are nitrocellulose and PVDF (polyvinylidine difluoride). Nitrocellulose was the first support used for Western blotting, and is still widely used. It probably binds proteins through hydrophobic interactions; the binding is blocked by oils. Nitrocellulose has a relatively high capacity for protein ( 50-100 µg/cm2 ), but it is also brittle and difficult to work with. In addition, the binding of proteins to the membrane has proven reversible under some circumstances, leading to sample loss and poorer detection limits.

Electrophoresis

One of the most straightforward applications of immunological detection is the ELISA, or enzyme linked immunosorbent assay (Figure 4.2.3d). In the simplest system, bound antigen is probed with antibodies which carry covalently attached enzyme molecules. Antibody binding immobilizes enzyme in the vicinity of the bound antigen, allowing detection of the antigen. Variations include a competition ELISA in which sample antigen is used to titrate the antibody from bound antigen. In this system, comparison of signal with signals from known antigen standards allows very accurate quantitation. In sandwich (capture) ELISAs, antibody is bound to a surface, and used to capture the antigen for detection by a second antibody. Sandwich ELISAs are extremely specific, because the antigen must react with 2 antibodies to be detected. ELISA signals may be chromogenic and interpreted by eye or spectrophotometer, or luminogenic and detected by a luminometer. Typically, ELISA’s are run in 96 well plates which are scanned (and sometimes processed) by automated devices.

PVDF is mechanically stronger than nitrocellulose. It binds proteins more tightly and has up to two fold higher capacity. Proteins immobilized on PVDF can be analyzed by microsequencing or amino acid analysis in addition to Western blotting. The PVC-like nature of this material gives it a resistance to mechanical and chemical stresses, and provides hydrophobic ”pockets” for denatured protein binding. For these reasons, PVDF has gained considerable popularity for Western blotting use. Once the protein has been transferred to the membrane, the membrane (or blot) is blocked to prevent unoccupied protein binding sites from nonspecifically immobilizing antibodies in the following steps. Blocking generally is carried out by incubating the blot in skim milk, BSA or some other complex mixture of proteins and surfactants such as National Diagnostics’ ProtoBlock (CL-252). The blocked blot is then ready for antibody probing.

Figure 4.2.3d The variations of ELISA (Enzyme Linked Immunosorbent Assay). Top: Basic ELISA, in which antigen is bound to a surface and probed with enzyme linked antibody, providing semi-quantitative information. Middle: Competitive ELISA. Purified antigen is bound to the surface. Probing is carried out in the presence of samples or dilutions of free antigen (standards). The free antigen competes with the bound antigen, reducing the amount of antibody bound. Comparison between samples and standards yields quantitative information. Bottom: Sandwich ELISA, using one antibody to capture the antigen and another to detect it, resulting in increased specificity.

Western Blotting

Proteins can also be detected immunologically following electrophoresis, a technique known as Western Blotting. This method relies on the fact that most epitopes (sites recognized by antibodies, generally comprising several amino acids) are still recognizable following denaturing of the protein with SDS and binding to the surface of a membrane. The two primary advantages of Western Blotting are sensitivity and specificity. Silver staining may detect 10ng of protein, and it will detect all proteins in a given sample. Western blotting can detect as little as 0.1ng of protein, and it will selectively detect only the protein of interest. Thus a complex mixture containing only traces of the desired protein may be analyzed accurately with this technique.

Gel

In order to achieve maximum sensitivity most Western blots are probed in two steps, using a “primary” and “secondary” antibody (Figure 4.2.3f). The primary antibody detects the protein of interest. If this antibody is “polyclonal” (that is, derived from an immunized animal, and therefore containing antibodies to multiple epitopes on the same protein) many antibodies will bind to each protein molecule. The secondary antibody is directed against the primary antibody, and is generally polyclonal, so several 2° antibodies will bind each 1° antibody. The result is an amplification, in which many 2° antibodies are bound to the site of each protein. The 2° antibody carries the enzyme label. Following binding and washing, detection reagents are applied to the gel to visualize the band(s) detected. Chromogenic substrates cause colored bands to appear on the white blot. If luminescent systems are used, the blot is placed against a piece of X-ray film and exposed, typically for 1 - 10 minutes.

Membrane

Filter paper Foam pads

Negative plate

Rubber bands Positive plate

er Buff ber cham

Figure 4.2.3e The Western Blotting apparatus. Proteins are electrophoresed from the gel to the membrane.

Figure 4.2.3f In western blotting, use of polyclonal primary and secondary antibodies results in amplification of the signal.

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Electrophoresis Applications - Post Electrophoretic Analysis

Protocol 4.2.3a Western Blotting

Applications

Electrophoresis

ELECTROPHORESIS Prepare and run an SDS PAGE gel as described in Protocol 3.1.2a. Select a gel percent which will give the best resolution for the size of antigen being analyzed (if known). If the size is not known, a 12% gel is a good starting point. Load enough protein to provide 0.1-1ng of antigen per well. It is generally advantageous to load serial dilutions of sample, so as to ensure that one lane at least will fall in the optimal range for detection. If desired, use prestained markers, such as National Diagnostics’ ProtoMarkers, to monitor transfer efficiency. TRANSFER Upon completion of the run, the gel must be transferred onto a blotting membrane. This can be accomplished by semi-dry transfer or wet transfer. Both are electrophoretic transfers, and require equipment designed for that purpose. The procedures outlined below are intended as general outlines. For best results and to ensure safety, follow the equipment manufacturer’s instructions for this phase. SEMI-DRY BLOTTING 1. Rinse electrode plates with deionized H2O. 2. Cut 6 sheets of Whatman 3MM paper and 1 sheet of blotting membrane to the size of the gel, or slightly smaller. 3. Wet the membrane. Soak nitrocellulose in deionized H2O for 3 minutes. Soak PVDF in methanol for 1 minute. Transfer membrane to transfer buffer and soak for 3 minutes. Transfer buffer: 0.025M Tris base 0.192M Glycine 20% MeOH

4. Assemble transfer “sandwich” in order:

5. Assemble sandwich clamps and support pads as per equipment manufacturer’s instructions. 6. Place sandwich in transfer tank with membrane side closest to the positive electrode (Red lead). 7. Add cold transfer buffer, and initiate cooling procedure. 8. Apply voltage: This parameter is entirely dependent upon the apparatus used. Large format gels may require 50 - 75V, 5 - 15 hours. Minigels can be blotted in 1 hour @ 50 - 100V in some systems. POST TRANSFER It is prudent to mark the blot in a permanent way for orientation, by notching or clipping a corner. If prestained markers were used, their positions should be marked in pencil or an alcohol indelible ink. Often well positions can be distinguished and marked immediately after transfer. Notes: Nitrocellulose membranes may be air dried prior to further processing. It has been reported that this improves protein retention on the blot. After transfer, Coomassie staining of the gel (Protocol 4.2.2c) can give information about the efficiency of transfer. 1. Stain the blot as described in Protocol 4.2.2g, and mark the positions of well and markers. If prestained markers were used, this step may be skipped. 2. At this point it is very helpful to spot diluted primary and secondary antibody on an unused area on the blot. This can be invaluable for troubleshooting. If a blot fails (no bands are detected) the antibody spots can be interpreted as follows: Visible: 1° & 2°- 2° antibody functioning well - label okay, 1° antibody may have failed. 2° only- 2° antibody failed to bind 1° no spots- Label enzyme denatured - remake 2° antibody dilution.

ASSEMBLE THE TRANSFER STACK 1. On the lower plate (positive, red lead), place in order: 3 sheets of 3MM (precut and wetted with transfer buffer) Transfer membrane Gel 3 sheets 3MM (precut and wetted with transfer buffer) Remove any air bubbles by rolling over each layer as placed with a 10ml pipette. Be careful not to disturb the stack, or to let the gel stick to the pipette. Roll gel after placing first upper layer of paper, if desired. Note that bubbles trapped in the stack will distort current flow, leading to lateral band displacements and failure of bands to transfer.

2. Check to be sure that no portion of the upper paper stack contacts the paper or the electrode underneath the gel. Contacts between upper and lower stacks will short circuit the current, distorting the transfer. In some systems, parafilm or plastic wrap may be arranged around the gel to prevent this short circuiting from occurring. Check the instructions. 3. Place the upper (negative, black lead) electrode plate on top of the stack, and apply current. Consult apparatus instructions; typical conditions are 1 hour @ 0.8 mA/cm2. Over-transfer may dry the gel and drive proteins through a Nitrocellulose membrane. WET TRANSFER 1. Cut 2 sheets of Whatman 3MM paper and 1 sheet of transfer membrane to the size of the gel.

BLOCKING A variety of blocking reagents are available. It is worthwhile to optimize blocking procedures, as this step determines the background level of the blot, and hence the detection limit. The most universal blocking agents contain mixtures of proteins and surfactants. This combination provides good to excellent blocking on most membranes. 1. Blocking Solutions

a. ProtoBlock: Dissolve 10g of Reagent B in 170 ml di water. Add 20 ml of Reagent A.

b. Tween/milk: Dissolve 50g nonfat dry milk and 2 g Tween 20 in 1L PBS. If product is to be stored for > 1 day, add 0.2 g NaN3 (CAU- TION - TOXIC!)

2. Blocking Procedure:

a. Immerse blot in blocking agent with agitation (i.e. a shaking or rock ing table) for 1 - 2 hours at room temperature.

b. Rinse blot in PBS + 0.2% Tween 20 twice for 5 minutes each.

Blot is now ready for antibody hybridization

2. Wet the membrane: Soak Nitrocellulose in water for 2 minutes. Soak PVDF in methanol for 2 minutes.

ANTIBODY PROBING

3. Place membrane in transfer buffer (see “Semi-Dry Blotting” above)

1. Dilute 1° and 2° antibodies into PBS + 0.2% Tween (PBST).

continued

Filter paper sheet Gel Membrane Filter paper sheet

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continued

Electrophoresis Applications - Post Electrophoretic Analysis

chemiluminescent detection reagent

Note: Including blocking reagent at 0.05 - 0.1X in the antibody solutions will decrease background without significantly lowering band intensity.

a. Mix equal volumes A and B reagents. Use 0.1-0.2ml/cm2 of membrane. Allow combined solution to come to room temperature.

The optimal dilution must be determined for each antibody. The 2° antibody may be tested on dots of 1° antibody. Dilutions can range from 1:100 to 1:10,000.

2. Incubate blocked, washed blot with 1° antibody for at least 1 hour at room temperature with agitation.

c. Wrap blot in plastic wrap, and place in a film cassette.

4. Incubate blot with 2° antibody for 0.5 - 1 hour. 5. Wash blot four times for five minutes each with PBST. 6. Transfer blot to detection reagent.

Notes: The extreme sensitivity of ProtoGlow ECL can lead to high backgrounds, in which the entire blot appears as a black image on the film, even at short (<1 minute) exposures. Background can be lowered by rinsing the blot for 1 minute in PBST after removal from the detection solution. Light emission is stable for hours. Multiple exposures may be taken over this period.

STRIPPING

DETECTION The most popular antibody labels are isotopic (125I), HRP and Alkaline Phosphatase. 125I labeling is straightforward, and gives consistent and quantifiable results. Its drawbacks are the hazards and inconvenience which radioactive isotopes bring into the lab. Detection of 125I requires that the blot be placed against X-ray film (protocol 4.1.3a). Upon development, the film will show bands corresponding to the position and intensity of detected antibody band.

Stripping PVDF blots with ProtoLift Western Stripping Buffer: ProtoLift Western Stripping Buffer is straightforward to use. The procedure is outlined below. CAUTION: This procedure requires the use of mercaptoethanol, a hazardous substance. Work in a fume hood.

A) 0.5g NitroBlue Tetrazolium in 10 ml 70% Dimethylformamide B) 0.5g BCIP in 10 ml 100% DMF C) 100mM NaCl 10mM MgCl2 100mM Tris pH 9.5

1. Probe the membrane using your preferred protocol. (do not allow the membrane to dry before stripping) 2. Rinse the membrane in two washes of PBS or TBS. 3. Prepare a working ProtoLift Western Stripping Buffer solution by adding 2-mercaptoethanol to 0.7% (v/v). To prepare 10 ml of working solution, add 70μl mercaptoethanol to 10 ml ProtoLift stock solution. 4. Place the membrane in a dish and add enough working solution to completely immerse the membrane. Alternatively the membrane can be sealed in a plastic bag with the working solution. 5. Incubate at room temperature for 10 minutes with shaking. PVDF membranes will become transparent in the solution. This is normal and the membrane will return to its regular appearance after the stripping solution is removed. 6. Wash the membrane three times for 10 minutes each with large volumes of PBS or TBS to remove the stripping solution and reducing agent.

1. To prepare substrate solution: mix 100µl A, 15ml C, then add 50µl B

The blot is now ready to be reprobed.

2. Submerge blot (up to 150 cm2) in 15ml substrate solution. Scale up the amount of solution for larger membranes. Incubate with shaking at room temperature until desired band intensity and contrast is achieved (typically 30 minutes) depending on antibody and label activity.

Stripping Nitrocellulose Blots

A variety of substrates are available for both alkaline phosphatase and HRP. Protocols are given below for the most commonly used. ALKALINE PHOSPHATASE: BCIP/NBT Stock solutions (stable for up to 1 year):

3. Stop development in PBS + 20mM EDTA Note: This stop reagent works for CIP or other eukaryotic phosphatases. It does not stop Bacterial Alkaline Phosphatase.

Nitrocellulose blots may be stripped and reprobed, albeit with some loss of sensitivity. Stripping generally involves the use of reducing agents such as 2-mercaptoethanol to cleave the disulfide bonds which hold the antibody probes together.

HORSERADISH PEROXIDASE

Stripping solution: 2g SDS 750 µl 2-mercaptoethanol 100ml 65mM Tris HCl pH6.8

1. Chromogenic detection with DiaminoBenzidine (DAB)

Incubate blot in stripping solution 60 minutes @ 50°C

Applications

d. Expose blot to X-ray film for 1 - 5 minutes, and develop film as usual.

3. Wash blot four times for five minutes each with PBST.

Electrophoresis

b. Immerse blot in combined A & B reagents at room temperature with shaking 1 minute.

(Caution: DAB is a carcinogen - avoid skin contact. Decontaminate spent material with bleach prior to disposal)

a. Make fresh detection solution: 9mg DAB Tetrahydrochloride 7ml 100mM Tris pH 7.6 1.5ml 0.3% NiCl2 (CoCl2 may be substituted) 6ml H2O Filter through Whatman 1 paper Add 15µl 30% H2O2 (or 150µl 3%)

Notes: This solution contains high concentrations of 2-mercaptoethanol: use and heat only in hood. Stripping time and temperature given are typical. Optimal values must be determined for each antibody/Antigen combination.

b. Immerse blot in detection solution (up to 150 cm2/15ml) and shake at room temperature until desired band intensity and contrast are achieved. (Typically <10 minutes)

c. Stop reaction by rinsing blot with agitation in PBS.

Note: DAB development with HRP is much more rapid than the AP/BCIP system. Also, because the stop solution is simply rinsing away substrate, the reaction may continue for a time after “stopping”. Development should be taken only up to the point where bands are acceptable and no background has yet appeared.

2. Chemiluminescent Detection using National Diagnostics’ ProtoGlow ECL continued

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis - Troubleshooting

Matrix Related Problems

Troubleshooting

Electrophoresis

Problems with the matrix will manifest as smeared bands and/or loss of resolution. Note that most “total failures” of electrophoresis are not matrix related (see “D”- Buffer related problems)

1) Pore size: This is determined by the amount of matrix and cross-linker used; suggested ranges are given on page 95. Using the wrong formulation will result in the region of interest running off of the gel, or failing to enter the gel sufficiently to resolve. Larger pore sizes also allow more diffusion, resulting in broader bands. 2) Polymerization: Gels which take longer than usual to polymerize will generally give broad and distorted bands due to local variations in gel quality. Polymerization of the gel is a chain reaction, initiated by ammonium persulfate (APS) and TEMED. The extension of initiated chains is inhibited by oxygen. APS and TEMED must be fresh for consistent results. APS should “crackle” when dissolving. TEMED should be clear, with no yellow color. Use of old reagents will lead to incomplete polymerization, which is further inhibited by low levels of dissolved oxygen. Degassing will remove O2, giving better polymerization, but it is not a substitute for fresh reagents. Cold temperatures will also slow polymerization- cast gels at room temperature. 3) Well Formation (sharks-tooth comb): The bottom of the well must be smooth and flat. Remove the comb after casting slowly, under buffer. Reinsert the comb carefully, so as not to over-penetrate the gel surface. Do not withdraw the comb once inserted- well distortions and leakage will result. 4) Immobilized charges: Acrylamide is subject to oxidation, producing acrylic acid. The immobilization of acrylic acid in the matrix generates a reverse flow of water (electroendosmosis), and allows DNA to interact with the matrix which leads to band broadening. Use only fresh acrylamide solutions within their shelf life which have been properly stored.

Sample Preparation

Degradation of samples leads to smeared or doubled bands. Overloading will cause bands to smear and will alter band positions.

1) Nuclease degradation: Exonucleases will cause bands to smear down, endonucleases will generate new bands. Keep samples cold, include EDTA where practical. 2) Denaturation: Insufficient heating may allow retention of residual secondary structure which causes bands to migrate at spurious molecular weights and may cause doublets. 3) Overloading: Loading of more than 10 micrograms per lane of DNA will cause an upward smearing of the bands. 4) Urea in wells: The urea in the gel will diffuse into the wells and disrupt sample loading. Flush wells with running buffer just prior to loading.

T r ou b l eshooti ng De n a t uri n g DN A-PAGE Ge l s Symptom

Diagnosis

Smeared bands or distorted band pattern- dyes run normally (gel difficult to load)

Gel poorly polymerized (A-2), gel overloaded (B-3), nuclease in samples (B-1), salt in samples (D-1), or wells not flushed (B-4).

Smeared bands or distorted band pattern- dye run speed altered or dyes smeared

Buffer mismatch (D-1,3,4), glycerol in samples (D-5), run parameters not optimal (C-1), or fixed charges on gel (A-4).

“Smiling”- middle lanes run faster than outer lanes

Gel heating during run- check run parameters (C-1), buffer (D-3,4) and heat exchange system on gel apparatus (C-3).

Wavy bands

Poor gel polymerization (A-2) or upper edge of gel distorted (A-3).

Double bands

Samples not fully denatured (B-2), nuclease degradation (B-1), or insufficient pre-run (C-2).

Gel fails to polymerize

Ammonium Persulfate or TEMED too old (A-2), or too much oxygen in the gel solution (A-2).

Dye runs slowly, as a sharp band at the top of the gel

No buffer in gel (D-2).

Expected bands not seen

Nuclease digestion of samples (B-1) or wrong pore size used (A-1).

Run Conditions

The voltage and temperature maintained during the run can have marked effects on the results.

1) Electrical parameters: run gels at constant wattage (45-55 watts), to maintain the temperature above 50°C, which keeps the sample denatured. Too low a wattage allows the gel to cool to the point that the samples will begin to renature. Running at too high a voltage causes smearing and/or “smiling”. 2) Pre-Run: Gels must be pre-run for 30 min. to warm the gel to operating temperature. Failure to pre-run gives doublet bands. 3) Heat exchange: Heat must be conducted away from the gel to avoid thermal gradients in the gel, which cause “smiling” or other pattern distortions.

USA: 1-800-526-3867 EUROPE: 441 482 646022

Buffer Related Problems

Buffer problems are by far the most common cause of “total failure” of gels. A buffer imbalance will lead to changes in the voltage, current or wattage of the run.

1) Sample contains too much salt: Salt ions in the sample will migrate as a zone of high conductivity, low resistance and low voltage. The initial migration of the sample bands will be altered, resulting in skewed or broad bands, or aberrant band positions. High salt samples should be dialyzed or otherwise desalted before loading. 2) No buffer in gel: This creates a high resistance across the gel. With no ions to carry the current, the tracking dye “stacks” in the zone behind the slow moving buffer front. 3) Tank buffer incorrect: Tank buffer that is too concentrated or too dilute will lead to a salt discontinuity which migrates through the gel, distorting the band pattern with a “wave” effect. 4) Gel buffer incorrect: The gel provides most of the resistance to current flow- changes in the gel buffer will alter the electrical parameters of the run. Too concentrated a gel buffer will allow more current to flow, leading to more heat generation, with distortions such as smiling. Too dilute a gel buffer will increase resistance to current flow, which may actually sharpen the bands but generally slows the run and reduces resolution. 5) Glycerol containing samples: Glycerol forms complexes with the borate in TBE buffer, creating a zone of low conductivity which migrates slowly through the gel, creating a “wave” pattern. Use a non-glycerol loading buffer, or run gels in a borate free buffer such as TTE.

Electrophoresis - Troubleshooting

Matrix Related Problems

Problems with the matrix will manifest as smeared bands and/or loss of resolution. Note that most “total failures” of electrophoresis are not matrix related (see “D”- Buffer related problems)

3) Wells: Use care in removing the comb after casting; check to be sure that the gel is completely cooled. Twisting of the comb on removal will tear the walls of the well, allowing sample to seep into the tear, and generating distorted band shapes. Rapid removal of the comb will create a vacuum which will tear out the bottom of the wells, allowing sample to seep out before it can enter the gel. 4) Immobilized charges: Poor grades of agarose can carry sulfate groups which, under electrophoresis, will generate a reverse flow of water (electroendosmosis), and which will also interact with the sample. Both effects lead to band broadening. Use only fresh, high quality Agarose, with an EEO specification of <0.15.

1) Nuclease degradation: Exonucleases will cause bands to smear down, endonucleases will generate new bands. Keep samples cold, include EDTA where practical. 2) Overloading: Loading of more than 10 micrograms per lane of DNA will cause an upward smearing of the bands. 3) Dye overload: In some cases, bands will comigrate with the tracking dye. The dye will absorb the ethidium fluorescence during staining, obscuring the band. Soaking the gel in water or buffer for 1 hour will allow the dye to diffuse enough to see the bands.

Troubleshooting

2) Gel preparation: Agarose should be boiled only long enough to dissolve all crystals. Overboiling will weaken the matrix. Cool to 60°C before pouring to avoid damage to the gel mold. Allow the gel to cool gradually after pouring- rapid cooling will “trap” swirling and convection currents in the set gel, which can distort bands which pass through them. NOTE ON ALKALINE GELS: alkaline gel buffers will hydrolyze hot agarose solutions. Boil agarose for alkaline gels in water, cool, and then add alkaline buffer concentrate just before pouring the gel.

Degradation of samples leads to smeared or doubled bands. Overloading will cause bands to smear and will alter band positions.

Electrophoresis

1) Pore size: This is determined by the concentration and type of agarose used. Suggested ranges are given on page 95. Low concentration gels should be run in the cold room, they are fragile, and allow more sample diffusion. High concentration gels are friable (crumble easily), and will not allow large DNA molecules to enter the gel.

Sample Preparation

T r ou b l e sho ot i n g Ag a rose DN A Ge l s Symptom

Diagnosis

Smeared bands- dyes run normally

Gel overloaded (B-2), nuclease in samples (B-1), or excessive salt in samples (D-1).

Smeared bands- dye run speed altered or dyes smeared

Run parameters not optimal (C-1,2), buffer mismatch (D-1,3), or fixed charges on gel (A-4).

“Smiling”- middle lanes run faster than outer lanes

Gel heating during run-check run parameters (C-1,2) and buffer (D-3).

Wavy bands

Gel cooled too quickly after pouring (A-2) or wells damaged (A-3).

Double bands

Nuclease degradation (B-1), gel run too fast (C-1), or buffer mismatch (D-3).

Gel fails to set up

Alkaline buffer hydrolyzed matrix (A-2). Agarose concentration too low (A-1).

Dye runs slowly, as a sharp band at the top of the gel

No buffer in gel (D-2).

Expected bands not seen

Nuclease digestion of samples (B-1), wrong percentage gel used (A-1), voltage too high (C-1), or dye overload (B-3).

Run Conditions

The voltage and temperature maintained during the run can have marked effects on the results.

1) Electrical parameters: Most gels are meant to be run at 5-15V/cm. Running at too low a voltage (too slow) allows the bands to diffuse too much before the run is finished. Too high a voltage causes heating which can melt the gel. Increasing the voltage also selectively increases the mobility of larger DNA molecules, compressing the band pattern. 2) Temperature: In general, gels should be run as cold as is convenient. This is particularly true for low-melting or low percentage gels, which are fragile at room temperature.

Buffer Related Problems

Buffer problems are by far the most common cause of “total failure” of gels. A buffer imbalance will lead to changes in the voltage, current or wattage of the run.

1) Sample contains too much salt: Salt ions in the sample will migrate as a zone of high conductivity, low resistance and low voltage. The initial migration of the bands will be altered, resulting in skewed or broad bands, or aberrant band positions. High salt samples should be dialyzed or otherwise desalted before loading. 2) No buffer in gel: Failure to add buffer to the gel creates a high resistance across the gel, with no ions to carry the current. The tracking dye “stacks” in the zone just behind the buffer front, and migrates slowly into the gel. 3) Gel or tank buffer incorrect: A mismatch between gel and tank buffers will lead to a salt discontinuity which migrates through the gel, distorting the band pattern with a “wave” effect. This can also cause bands to migrate at an angle to the vertical, causing the bands to look broad when viewed from directly above. In some cases, such bands will appear to be doublets.

USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis - Troubleshooting

Matrix Related Problems

Troubleshooting

Electrophoresis

Problems with the matrix will manifest as smeared bands and/or loss of resolution. Note that most “total failures” of electrophoresis are not matrix related (see “D”- Buffer related problems)

1) Pore size: This is determined by the amount of matrix and cross-linker used; suggested ranges are given on page 95. Too small of a pore size will prevent proteins of interest from moving far enough into the resolving gel to be resolved. Too large of a pore size will allow smaller proteins to run with the SDS front, creating a large, intense protein band containing many unresolved proteins, running just behind or with the tracking dye. 2) Polymerization: Polymerization of the gel is a chain reaction, initiated by ammonium persulfate(APS) and TEMED. The extension of initiated chains is inhibited by oxygen. APS and TEMED must be fresh for consistent results. APS should “crackle” when dissolving. TEMED should be clear, with no yellow color. Use of old reagents will lead to incomplete polymerization, which is easily inhibited by low levels of dissolved oxygen. Such gels will take longer than usual to polymerize, and will give broad and distorted bands due to local variations in gel quality. Degassing will remove O2, giving better polymerization, but it is not a substitute for fresh reagents. 3) Stacking gel: This gel sharpens the protein zone before it enters the resolving gel. A straight sharp interface is critical to good resolution- overlay the resolving gel with water saturated n-butanol for best results. 4) Immobilized charges: Acrylamide is subject to oxidation, producing acrylic acid. The immobilization of acrylic acid in the matrix generates a flow of water (electroendosmosis), and allows the proteins to interact with the matrix, which leads to band broadening. Use only fresh acrylamide solutions, within their shelf life, which have been properly stored.

Sample Preparation

Degradation of samples leads to smeared or doubled bands. Overloading will cause bands to smear and will alter band positions.

1) Proteolysis/Oxidation: Proteases are active in the sample buffer- keep the samples cold prior to denaturing and loading. Add mercaptoethanol or DTT to the sample buffer if oxidation is suspected. Proteolysis can cause spurious bands or a downward smearing. 2) Denaturation: Insufficient heating may allow retention of residual secondary structure which will cause bands to migrate at spurious molecular weights, and may cause doublets. 3) Overloading: Loading of more than 40 micrograms per lane of protein will cause an upward smearing of the bands.

T r ou b l eshooti n g De n a t uri n g Prot e i n Ge l s Symptom

Diagnosis

Smeared bands- dyes run normally

Gel poorly polymerized (A-2), gel overloaded (B-3), protease in samples (B-1), or excessive salt in samples (D-1).

Smeared bands- dye run speed altered or dyes smeared

Run parameters not optimal (C-1), buffer mismatch (D-1,3,4), or fixed charges on gel (A-4).

“Smiling”- middle lanes run faster than outer lanes

Gel heating during run - Check run parameters (C-1), buffer (D-3,4) and heat exchange system on gel apparatus (C-2).

Wavy bands

Poor interface between stacking and resolving gels (A-3). Poor gel polymerization (A-2).

Double bands

Protease degradation or sample oxidation (B-1), or samples not fully denatured (B-2).

Gel fails to polymerize

Ammonium Persulfate or TEMED too old (A-2). Too much oxygen in the gel solution (A-2).

Dye runs slowly, as a sharp band at the top of the gel

No buffer in gel (D-2).

Expected bands not seen

Protease digestion of samples (B-1) or wrong pore size used (A-1).

Run Conditions

The voltage and temperature maintained during the run can have marked effects on the results.

1) Electrical parameters: Most gels are meant to be run at 5-20V/cm. Running at too low a voltage (too slow) allows the bands to diffuse too much before the run is finished. Running at too high a voltage causes the bands to smear, and will lead to overheating, which causes “smiling”. 2) Temperature: In general, gels should be run as cold as is convenient. If high voltages are used, a heat exchange system should be used. Overheating of the gel causes changes in the conductivity of the buffer, which leads to unpredictable results, often distortions of the band pattern, or band broadening.

USA: 1-800-526-3867 EUROPE: 441 482 646022

Buffer Related Problems

Buffer problems are by far the most common cause of “total failure” of gels. A buffer imbalance will lead to changes in the voltage, current or wattage of the run.

1) Sample contains too much salt: Salt ions in the sample will migrate as a zone of high conductivity, low resistance and low voltage. Until this zone migrates away from the sample, the migration of the protein bands will be altered, resulting in skewed or broad bands, or aberrant band positions. High salt samples should be dialyzed or otherwise desalted before loading. 2) No buffer in gel: Failure to add buffer to the gel creates a high resistance across the gel, with no ions to carry the current. The tracking dye “stacks” in the zone just behind the buffer front, and migrates slowly into the gel. 3) Tank buffer incorrect: The Laemmli buffer system uses a buffer discontinuity to “stack” the samples prior to separation; as a result it is more tolerant of minor buffer changes than a continuous system. Use of too concentrated or too dilute tank buffer will lead to a salt discontinuity which migrates through the gel, distorting the band pattern with a “wave” effect. 4) Gel buffer incorrect: The gel provides most of the resistance to current flow- changes in the gel buffer will alter the electrical parameters of the run. Too concentrated a gel buffer will allow more current to flow, leading to more heat generation, with distortions such as smiling. Too dilute a gel buffer will increase resistance to current flow, which may actually sharpen the bands but generally slows the run and reduces resolution.

Electrophoresis - Useful Information

Useful Information for Electrophoresis Effective Range of Separation of DNAs and Dye Co-Migration in Denaturing Polyacrylamide Gels

Effective Range of Separation of DNAs and Dye Co-Migration in Native Polyacrylamide Gels

19:1 Acrylamide/Bis-Acrylamide

29:1 Acrylamide/Bis-Acrylamide

Size Range (bp)

1000-2000

450

>250

155

70-450

240

60-250

110

Gel %

60-400

160

40-120

50-300

120

20-60

40-200

10-50

Range of Separation in Agarose Gels Gel %

Size Range (bp)

0.3

5000-60,000

0.6

1000-20,000

0.7

800-10,000

0.9

500-7000

1.2

400-6000

1.5

200-3000

2.0

100-2000

Useful Information

Xylene Cyanol Bromophenol Blue Xylene Cyanol (nucleotides) (nucleotides)(nucleotides)

Size Range (bp)

Electrophoresis

Xylene Cyanole Bromophenol Xylene Blue Cyanole (nucleotides) (nucleotides)(nucleotides)

Gel %

The Genetic Code The sieving characteristics of different types of agarose vary considerably. A more complete discussion can be found in Section 2.4.1 (page 87).

A A A

G T

C T

Lys Lys Asn Asn Arg Arg Ser Ser Ile Met Ile

Ile Thr Thr Thr Thr

G A A

G T

C T

Glu Glu Asp Asp Gly Gly Gly Gly Val Val Val Val Ala Ala Ala Ala

Range of Separation of Proteins in SDS-PAGE Gel %

Size Range (kd)

60-200

40-140

20-80

15-70

10-50

Stop Stop

G C

Tyr Tyr

A Stop

C T

Trp Cys Cys Leu Leu Phe Phe Ser Ser Ser Ser

C A A

G T

C T

Gln Gln His His Arg Arg Arg Arg Leu Leu Leu Leu Pro Pro Pro Pro

DNA Data

Base pairs per turn (B form): 10 1 microgram of 1000bp DNA=1.51pmoles

Melting Point Calculations:

TmDNA:RNA = 79.8° C + 18.5 (log10[Na]) + 0.58 (%GC)+ 11.8 (%GC)2 + 0.50 (% Formamide) + (820/length) TmDNA:DNA = 81.5°C - 16.6(log10([Na])+0.41(%GC) - 0.63(%Formamide) - (600/length)

avg MW of a base pair=650 Tm decreases by ~ 1° C for every 1% increase in mismatches. Tm decreases by ~ 0.5° C for every increase of 1% in formamide.

Protein Data

Average MW of an amino acid:110 daltons grams SDS bound per gram of protein: 1.2 (average) A280 1mg/ml solution: 0.4-1.5 (range can be as much as 0.0-2.65)

UV Absorbance of DNA and RNA Nucleic Acid

µg/ml to give an A260=1.0

A260/A280 of pure material

ds DNA ss DNA RNA

50 37 40

1.8 1.8-1.9 2.0

Range of separation tables adapted from Sambrook, J., Fritsch., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory. USA: 1-800-526-3867 EUROPE: 441 482 646022

Electrophoresis - Suggested Reading

Electrophoresis

Suggested Reading

Wieslander L. (1979) A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal. Biochem. 98 305.

Bloom, H., Beier, H, and Gross, H.S. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8 93-99.

Blotting Nucleic Acids - Northerns and Southerns

Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. PNAS(USA) 77 5201. Reed, K.C. and Mann D.C. (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13 7207. Casey, J. and Davidson, N. (1977) Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 4 1539-1552 Southern, E.M. (1975) Detection of Specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98 503-517. Meinkoth, J. and Wahl, G. (1984) Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138 267-284,

Post-Electrophoretic Protein Detection

Machenko, Gennady (ed) (1994) Detection of Enzymes on Electrophoretic Gels: A Handbook. CRC Press.

Staining Proteins in Gels

Wilson, C.M. (1979) Studies and critique of amido black 10B, coomassie blue R, and fast green FCF as stains for proteins after polyacrylamide gel electrophoresis. Anal. Biochem. 96 263-278. Blakesly, R.W. and Boezi, J.A. (1977) A new staining technique for proteins in polyacrylamide gels using coomassie brilliant blue G250. Anal. Biochem. 82 580-582.

Silver Staining Proteins

Bloom, H., Beier, H, and Gross, H.S. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8 93-99. Merril, C.R., Goldman, D. and Van Keuren, M.L. (1984) Gel protein stains: silver stain. Meth. Enz. 104 441-447.

Immunological Detection of Proteins (Western Blotting)

Burnette, W.H. (1981) Western blotting: electrophoretic transfer of proteins from SDS-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112 195-203. Bers, G. and Garfin, D. (1985) Protein and nucleic acid blotting and immunobiochemical detection . Biotechniques 3 276-288. Ramlau, J. (1987) Use of secondary antibodies for visualization of bound primary reagents in blotting procedures. Electrophoresis 8 398-402. Towbin, H. Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. PNAS(USA) 76 4350-4354.

Alkaline Phosphatase

Moe, D. and Kirkeby, S. (1982) Evaluation of the indoxyl-tetrazolium method for measurement of alkaline phosphatase activity. Cell. and Mol. Biol. 28 555-558.

Horseradish Peroxidase

Graham, R.C. and Karnovsky, M.J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14 291-302.

Enzyme Linked Immunosorbent Assay (ELISA)

Engvall, E., and Perlman, P. (1971) Enzyme linked immunosorbent assay (ELISA): quantitative assay of immunoglobulin G. Immunochemistry 8 871-879.

Misra, H.P. and Fridovish, I. (1977) Arch. Biochem. Biophys. 183 511-515. Gregory, E.M. and Fridovich, I. (1974) Anal. Biochem. 58 57-62.

Two Dimensional Electrophoresis

Oâ&#x20AC;&#x2122;Farrell, P.H. (1975) High-resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250 4007-4021.

USA: 1-800-526-3867 EUROPE: 441 482 646022

Histology Products - Clearing Agents

Histology

Clearing Agents

Histological Clearing Agents National Diagnostics has a history of innovation in providing alternatives to the use of hazardous chemicals in the laboratory. Because xylene is a serious health risk for technicians working in histology, cytology, anatomy and pathology laboratories, National Diagnostics offers a range of solvent substitutes that eliminate the hazards associated with xylene without compromising performance. Our clearing agents ensure both good results and safe practice.

Histo-Clear

l l l l l

Rapid Clearing without the Toxic Hazards of Xylene Biodegradable Greatly Reduces Disposal Costs Superior Results with Lower Citrus Odor Natural Product with Food Grade Rating World-Wide

The use of Histo-Clear in the laboratory means no longer having to breathe xylene when preparing histological sections. Distilled from oranges, carefully purified, and stabilized, Histo-Clear is one of the safest clearing agents available.

APP L IC A TION Processing Fixed Tissue - Clearing.............................................. 111

Histo-Clear makes histology

The Safety of Histo-Clear

safer and improves results.

The Hazards of Xylene Exposure to xylene may cause systemic toxicity including adverse effects to the kidney, liver, brain, blood, spleen, fetus and central nervous system. Repeated and prolonged occupational exposure has been associated with permanent brain and nervous system damage (sometimes referred to as solvent or painter’s syndrome). Xylene may cause adverse reproductive and/ or developmental effects. Pregnant women may be at an increased risk from exposure. Preexisting medical conditions which may be aggravated by exposure include disorders of the skin, eye, heart, kidney, liver, blood, respiratory system, neurological and hemopoietic organs.

100

USA: 1-800-526-3867 EUROPE: 441 482 646022

Histo-Clear not only improves safety but also results. Histo-Clear leaves tissue less hard and brittle than xylene, facilitating the cutting of thin sections and prolonging microtome blade life. Nuclear morphology is rendered in fine detail. Histo-Clear enhances the clarity and vibrance of acidophilic stains and improves staining of Harris’ Hematoxylin with a brighter Eosin background. Histo-Clear can directly substitute for xylene and yields excellent results in automated tissue processing.

Product Name

Cat. No.

Size

Histo-Clear HS-200 1 L 1 gal (1-3) 1 gal (4+) 5 gal 55 gal Mounting Media Histomount [pg 107] HS-103 100 ml (1-3) 100 ml (4+) 450 ml (1-3) 450 ml (4+) Hydromount [pg 107] HS-106 100 ml (1-3) 100 ml (4+) Omnimount HS-110 100 ml (1-3) 100 ml (4+)

Histology Products - Clearing Agents

His to-Cle a r II ®

l l

APP L IC A TION Processing Fixed Tissue - Clearing...............................................111

Low Hazard Biodegradable Reduced Citrus Odor

Cat. No.

Size

Histo-Clear II HS-202 1 gal (1-3) 1 gal (4+) 5 gal 55 gal

His tosol

APP L IC A TION Processing Fixed Tissue - Clearing.............................................. 111

Histology

Product Name

Clearing Agents

Histo-Clear II, like its predecessor Histo-Clear, is a safer histological clearing agent that leads to the production of high-quality tissue slides. Histo-Clear II is nontoxic and completely biodegradable, thus reducing disposal costs. Histo-Clear II has a greatly reduced citrus odor compared to other citrus-based histological clearing agents. Histo-Clear II may be substituted for toluene and xylene with minimal change in protocol.

Histosol is the original high flash point (114˚F TCC) histological clearing agent. It is intended to be used as a replacement for xylene where the hazards associated with aromatic hydrocarbon vapors are to be reduced. Museum-quality tissue slides can be prepared with Histosol without change in protocol or procedure. Histosol is manufactured from petrochemical products and is miscible in all proportions with ethanol, isopropanol, and t-butanol. It is also miscible with all paraffin-based tissue embedding media and all permanent mounting media. Product Name

Cat. No.

Size

Histosol HS-100 1 gal 5 gal

Histological Grade Reagent Ethanol

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Stringently Controlled Quality and Batch Consistency No Harmful Ketones Virtually No Residual Water 95% Ethanol / 5% Methanol

High quality ethanol is essential for excellent results in paraffin embedding technique and related methods. National Diagnostics Reagent Ethanol matches the demands of currrent histology and cytology practice. Stringently controlled specifications and intensive quality testing ensure that no residual water or chemical impurities are present to interfere with deparaffinizing, staining or mounting. The use of National Diagnostics’ Histological Grade Ethanol in tandem with National Diagnostics Histo-Clear—our low toxicity clearing agent—ensures excellent tissue processing results. Product Name

Cat. No.

Size

Reagent Ethanol (denatured) HS-300 1 L 4 L 20 L

USA: 1-800-526-3867 EUROPE: 441 482 646022

101

Histology Products - Tissue Preparation

Tissue Preparation

Histology

Tissue Preparation

Whether you need to decalcify bone or to fix tissue to preserve fine structure, National Diagnostics has a product that will help. Wherever possible our products are designed to reduce hazards to the operator without compromising performance.

Calci-Clear Calci-Clear Rapid 速TM

Calci-Clear may be used to decalcify any tissue type. This solution of efficient chelating and sequestering agents removes and binds all calcium and other metal ions under treatment. Interference with subsequent staining is reduced while tissue distortion and structure loss is minimized. Calci-Clear Rapid is a high-speed decalcifier which permits the processing of tissue samples in considerably less time than with other decalcifiers. Tissue distortion and structure loss are minimized while excellent staining characteristics are maintained. Product Name

APPL IC ATIO N Decalcifying Tissue Samples .......................................................111

102

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Cat. No.

Size

Calci-Clear HS-104 1 quart 1 gal. 5 gal. Calci-Clear Rapid HS-105 1 quart 1 gal. 5 gal.

Histology Products - Tissue Preparation

Mirsky’s Fixative

l l l l l

Histology

Fixing Tissue Samples................................................................. 109

Mirsky’s Fixative is a superior fixing agent for use in immunohistological and immunocytological staining protocols. Mirsky’s Fixative does not contain formaldehyde or glutaraldehyde. Therefore, it has considerably reduced toxicity and virtually no odor. Mirsky’s Fixative is neutral, buffered, and isotonic (308 milliosmol). Additionally, Mirsky’s Fixative does not contain toxic or hazardous buffers such as cacodylate or barbital, and can be safely used for a wide variety of tissue samples. With Mirsky’s Fixative, hardening or shrinkage of tissue is considerably reduced.

Tissue Preparation

AP P L IC ATIO N

Saponin/Glyoxylate Fixative Preserves Immunohistological Activity Contains No Formaldehyde Fast-Acting No Toxic Buffers

Double reactive sites afford excellent crosslinking properties while maintaining sample enzyme activity. Therefore, samples processed in Mirsky’s Fixative for light microscopy can subsequently be used in electron microscopy procedures. Special buffer systems may be used in place of the buffer provided. Method: Use as a replacement for formalin and/or glutaraldehyde fixatives in immunohistological and immunocytological staining protocols. Tissue size is unlimited as long as the sample is no thicker than 0.5 cm in at least one plane to assure uniformity of tissue penetration. Mirsky’s Fixative is crosslinking and will therefore retain cellular morphology better than non-crosslinking fixatives such as formaldehyde. It is expected that the gross visual appearance will be different than in formaldehyde and tissue may seem “raw.” This is due to double site binding of the fixative with a resultant reduction in tissue brittleness and shrinkage. Microscopic examination of tissue morphology will be noticeably improved. This material is intended to maintain enzyme and antibody activity. For best results, tissue sections of high digestive enzyme content should be thoroughly rinsed in saline solution before fixation (e.g. trypsin in intestinal samples). Once fixed, tissue may be retained in Mirsky’s Fixative and ethanol and is stable indefinitely. Mirsky’s Fixative is normally distributed as a concentrated two bottle system, although high throughput laboratories may be interested in the single bottle ready-to-use format. The two bottle system is comprised of Mirsky’s Fixative 10X Concentrate and Mirsky’s Fixative 10X Buffer. To reconstitute to working strength, add 1 part Mirsky’s Fixative 10X Buffer to 8 parts distilled or deionized water, mix thoroughly, then add 1 part Mirsky’s Fixative 10X Concentrate and mix again. The material is now ready for use. The two bottle system has a shelf life of 12 months while the ready-to-use format has a shelf life of 30 days. Product Name

Cat. No.

Size

Mirsky’s Fixative HS-102 200 ml system 2 liter system Mirsky’s Fixative (ready-to-use) HS-101 1 gal 5 gal

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Histology Products - Aldehyde Disposal

Safer Aldehyde Disposal Neutralin

速

Neutralizes Formaldehyde, Glutaraldehyde and other Aldehyde Solutions Convenient, Safer Disposal

Histology

Aldehyde Disposal

Neutralin is a convenient, cost-effective method for the disposal of hazardous formaldehyde, glutaraldehyde, and other aldehyde solutions. Neutralin converts hazardous aldehydes into a nonhazardous, noncorrosive, nontoxic polymer and water. The polymer produced is not a hazardous waste (as defined by United States Title 40 Code of Federal Regulations (40 CFR 261.24(a)). Neutralin reduces disposal costs and contributes to a safer work environment.

APPL IC ATIO N Safer Disposal of Aldehyde Waste........................................... 106

Simply pour used aldehyde solutions into the five gallon pail containing Neutralin and agitate. Neutralization occurs overnight without additional steps or decants. Neutralin is supplied with a Trace Aldehyde Detection Kit, which enables testing of the waste solutions to ensure the absence of residual aldehydes. Neutralin is prepacked and ready to receive five gallons of 4% formaldehyde (10% formalin) solutions.

Product Name

Cat. No.

Size

Neutralin HS-108 1 system

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Histology Products - Mounting Media

Mounting Media National Diagnostics mounting media have been laboratory standards for over twenty five years. They enable the researcher to produce museum quality slide preparations with crystal clear visibility.

APP L IC A TION Mounting Tissue Sections............................................................. 115

Histomount is the classic choice in synthetic mounting media. Histomount provides a permanent seal to store or ship slides with confidence.

Histomount is a pH neutral, UV stabilized preparation which retains its clarity and brilliance for years. Refractive index is matched to glass cover slips and slides, reducing chromatic aberration with any light source.

Histology

Histomount

Mounting Media

速

Histomount is effective with most clearing agents when used as a liquid cover slip or as a permanent mounting medium for traditional glass cover slipping. A dip stick providing an optimal amount of Histomount is provided with each 100 ml bottle.

Hydromount

Omnimount

Hydromount is the traditional choice whenever a nonfluorescing aqueous medium is needed. Hydromount is water-based and is suitable for mounting specimens that have been processed in water. Hydromount is effective for frozen sections, amyloid, and immunofluorescent staining procedures. Should it become necessary, Hydromount may be removed by soaking the slides in warm saline.

Specifically developed to provide compatibility with National Diagnostics clearing agent, Histo-Clear II, Omnimount combines outstanding optical characteristics with low fluorescence and exceptional durability. In addition to being the ideal partner for Histo-Clear II, Omnimount is a truly universal mounting medium, compatible with all common clearing agents: xylene, toluene, limonene, and petroleum derived products. The Omnimount solvent has a higher flash point and a lower toxicity than xylene based mountants, so Omnimount provides both a safer work environment and reduces shipping costs. Product Name

Cat. No.

Size

Histomount HS-103 100 ml (1-3) 100 ml (4+) 450 ml (1-3) 450 ml (4+) Hydromount HS-106 100 ml (1-3) 100 ml (4+) Omnimount HS-110 100 ml (1-3) 100 ml (4+)

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Histology Products - Histological Stains

Histological Stains Uniform and reliable, National Diagnostics’ stains are the highest possible quality. Their preparation entails special purification and conformity to high standards of performance.

Harris’ Hematoxylin

l l

Stains

Histology

H & E Staining Supplies

l l

Finest Control of the Ripening Process Longest Shelf Life Fewest Artifacts Reproducible Vibrant Staining

The most commonly used histological stain, hematoxylin is a natural compound extracted from a species of tree found in Mexico and the West Indies. The extracted compound is oxidized to produce hematein, the active staining component of the hematoxylin stain. The production of histological staining solutions is an art. Only a few laboratories are capable of developing a stain of reliable vibrance. At National Diagnostics we have gone one step further. Our proprietary methods achieve the finest control of the ripening process, resulting in the only commercially available Harris’ Hematoxylin with a full one year shelf-life. Product Name

Cat. No.

Size

Harris’ Hematoxylin HS-400 l liter

Eosin Solution

l l

1% Stock Solution Ready for Use in your Preferred Protocol

Product Name Eosin Solution

Scott’s Tapwater

l l l l

For Reliable Bluing of Hematoxylin Stains Ultra-Pure Reagents 0.1 Micron Filtration 18 megOhm Water

Scott’s Tapwater

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Size

HS-402 100 ml

Product Name

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Cat. No.

Size

HS-404 1 liter

Histology Products - Histological Stains

Bromophenol Blue Alcian Blue

HS-504

HS-603

10 g

Stains

Alcian Blue

Bromophenol Blue

Histology

Alcian Blue in conjunction with PAS (Mowry, 1956) is commonly used to differentiate acid and neutral mucopolysaccharides. It is also used with safranine for mast cell differentiation (Csaba, 1969.)

Dye for histones in alkaline & neutral buffer systems. Commonly used tracking dye in DNA, RNA and protein electrophoresis.

25 g

Fast Green FCF Amido Black

Recommended as a substitute for Light Green SF Yellowish in Masson’s trichrome as it is less likely to fade. It may be substituted Amido Black is a useful forensic stain typically used for enhancing for Light Green in many other procedures as well. latent prints contaminated with blood. Amido Black is very sen- Fast Green FCF HS-516 25 g sitive to the proteins found in blood. It leaves a black/blue stain and is usable on both porous and nonporous surfaces. Amido Black 10B

HS-601

25 g

Methyl Green Methyl Green in conjunction with Pyronin Y will differentiate RNA & DNA.

Basic Fuchsin

Methyl Green

HS-606

10 g

Basic Fuchsin is the main ingredient of Schiff’s reagent, a pH indicator, and can be used as stain for glycoproteins and mucopolysaccharide proteins in acidic pH systems. Usually used with Naphthol Blue Black as a post-stain color enhancer. Basic Fuchsin

HS-518

25 g

Methylene Blue Sensitive stain specific for DNA and RNA. Methylene Blue

Biebrich Scarlet

HS-525

25 g

Pyronin Y

Biebrich Scarlet can be used as a plasma stain instead of Acid Fuchsin in Masson’s trichrome. Pyronin Y in conjunction with Methyl Green will differentiate RNA Biebrich Scarlet HS-506 25 g & DNA. Pyronin Y

HS-607

5 g

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Histology Applications - Fundamental Histological Technique

Histology Fundamentals

1.1 FIXATION

Aldehyde Based Fixatives / Other Fixatives / Factors Affecting Fixation / Working Safely with Fixatives

1.2 DECALCIFICATION

1.5 ARTIFACTS IN HISTOLOGIC SECTIONS

1.6 STAINING

1.3 PROCESSING OF FIXED TISSUE

Histology

Fundamentals

Dehydration / Clearing / Embedding

Introduction / The Chemistry of Dyes / The Chemistry of Staining / Staining Procedures

1.7 MOUNTING

1.4 SECTIONING

Histology...Through the Looking Glass

istology is concerned with the relationships between life processes and the microscopic characteristics of tissues. In practice, histology involves the collection of tissues from the body and their examination under a microscope. The purpose of histological procedures is often to assist in the diagnosis of disease.

Thanks to the University of Bristol Department of Pathology and Microbiology.

Prior to microscopic examination, it is usually necessary to subject a tissue specimen to a series of processes in order to preserve the tissue and to highlight specific morphological characteristics. Although in practice there are many variations of histological technique, the most common means to prepare a slide for light microscopy is referred to as the paraffin technique. The paraffin technique consists of the steps of fixation, dehydration, clearing, embedding in paraffin, sectioning, rehydration, staining, and mounting.

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Histology Applications - Fundamental Histological Technique

1.1 Fixation To maintain the tissue in as lifelike a state as possible, tissue for analysis is usually placed directly into a fixative solution upon removal from the body. Fixation is normally carried out as soon as possible to prevent autolysis and to reduce possible infectivity. Several factors determine the choice of fixative for a given application. If morphological changes are known to take place rapidly after tissue collection, as with neural tissue, the speed of fixing action is extremely important. For large tissue samples, the rate of penetration of the fixative into the sample must also be considered. If immunological detection methods are to be used, a fixative which preserves protein structure is required. For Electron Microscopy, fixatives which do not precipitate proteins avoid artifacts invisible to light microscopy. The price of a fixative may also be a factor. The low cost of formaldehyde fixatives is one reason for their popularity. A partial list of fixatives, grouped by chemical type, would include:

Formaldehyde and glutaraldehyde are the most commonly used aldehyde fixatives. They work by forming cross-links both within and between proteins, particularly between lysine residues. Damage to the tertiary structure of the proteins occurs on a limited basis. Formalin (37% aqueous formaldehyde) is normally diluted 10 fold and neutrally buffered to make a working fixative solution consisting of 4% formaldehyde. The buffer prevents autolysis that occurs under acid conditions and also prevents the development of coloration of the tissue caused by “formalin pigment”.

Aldehyde fixative

Typical Applications

Buffered Formaldehyde

General use. The most widely used fixative.

Glutaraldehyde Electron Microscopy

Advantages

Disadvantages

Works well in a wide range of applications. Easily identifiable odor.

Toxic. Discolors tissues, may cause “formalin pigment” formation.

Good preservation of fine structure. Cross linking fixative.

Toxin, allergen, solution must be cold, and tissue should be <1mm thick.

Mirsky’s Fixative

General use. Electron microscopy. Glutaraldehyde replacement.

Excellent preservation of fine structure. Cross linking fixative. Antimicrobial.

Tissues must be sectioned to <3mm thickness.

Bouin’s Solution

Testis, GI tract, endocrine tissue.

Does not require removal of mercury deposits before staining.

Slow. Colors tissue yellow. May cause tissue shrinkage.

Picric Acid

Fixes histones and basic proteins.

Good glycogen preservation.

Explosive when dry. Shrinks tissue.

Zenker’s Fixative

Reticuloendothelial tissues: lymph nodes, spleen, thymus, bone marrow.

Excellent nuclear fixative. Enhances some stains.

Mercury deposits must be removed before staining.

95% Ethanol

Smears

Rapid. Easy. Low toxicity.

Causes denaturation and brittleness.

Histology

1.1.1 Aldehyde Fixatives

Fixative

Fundamentals

Aldehydes Mercurials Formaldehyde B-5 Glutaraldehyde Zenker’s Mirsky’s Fixative Alcohols Picrates Methanol Bouin’s solution Ethanol Oxidizing agents Osmium tetroxide Permanganate fixatives (potassium permanganate) Dichromate fixatives (potassium dichromate)

Common Fixatives

Table 1.1a Protocol 1.1.1a Figure 1.1.1a Aldehyde fixatives form crosslinks between proteins.

Microwave Fixation with Mirsky’s Fixative 1. Fix tissue blocks (<3mm on a side) in Mirsky’s fixative for 30 minutes at room temperature. 2. Microwave vials in a microwave processor.

1-3mm thick blocks: 15 minutes at 55°C < 1mm thick blocks: 4 minutes at 55°C

3. Transfer the block to 100% ethanol, and microwave for 4-8 minutes (increase time for thicker sections) at 67°C. Formaldehyde

Glutaraldehyde

Glutaraldehyde (normally used as a 2% buffered solution) causes deformation of alpha-helix structures in proteins, which limits its usefulness as a fixative for immunological stains. Glutaraldehyde is nonetheless a rapid fixative. As a result, it has become the standard for electron microscopy (Section 2.2). Recently glutaraldehyde has become identified as a powerful allergen. A maximum exposure limit of 0.05mg/m3 has been imposed on laboratories in the United Kingdom, sharply limiting the use of this fixative in that country. National Diagnostics’ Mirsky’s Fixative is an aldehyde based formula which is exceptionally good at preserving fine tissue structure and protein conformations. This fixative is an excellent replacement for Glutaraldehyde in EM applications. For light microscopy, Mirsky’s Fixative is especially recommended for immunoperoxidase techniques where formaldehyde can lead to the loss of epitopes. The use of microwave enhancement can speed the action of Mirsky’s Fixative greatly, allowing tissues to be embedded within a few hours.

4. Repeat ethanol/microwave treatment with fresh ethanol 2 times. 5. Microwave in preheated-paraffin for 7 minutes at 84°C

Products for Fixation Mirsky’s Fixative HS-102 (Concentrate and Buffer) Excellent for immunohistochemistry with enhanced enzyme and antibody activity. (pg.103)

Mirsky’s Fixative HS-101 (Ready-to-Use) Convenient, no-mixing formula. 30 day shelflife. (pg.103)

Neutralin HS-108 Improves safety by converting hazardous aldehyde waste into a nonhazardous polymer and water. (pg.104)

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Histology Applications - Fundamental Histological Technique

1.1.2 Other Fixatives Mercury Based Fixatives

Mercurials contain mercuric chloride. Their method of tissue fixation is poorly understood. While not penetrating tissue well and causing some tissue hardness, mercurials are fast and provide excellent nuclear detail. They are commonly used to fix hematopoietic and reticuloendothelial tissues.

HgCl2 Mercuric chloride

Alcohol Fixatives

Histology

Fundamentals

Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are sometimes used as fixatives. They tend not to be used routinely as they cause brittleness in tissue due to their dehydrating effect. However, they are very good for cytological smears because they act quickly and give good nuclear detail.

through tissue. Formalin and alcohol penetrate the fastest, glutaraldehyde the slowest, with mercurials and Mirsky’s Fixative falling between these extremes. Normally, slow rates of penetration are only a problem when dealing with whole organ perfusion, because thin tissue sections are easily permeated and not affected by this variable. Speed of fixation can be dramatically improved by the use of microwave protocols. The standard ratio of fixative to tissue volume is 10:1. Lower volumes can be used if frequent changes of the fixative are carried out to prevent exhaustion. Agitation will enhance the process by ensuring that fresh fixative solution is constantly washing over the surface of the tissue. Increasing the temperature will increase the speed of fixation, and hot formaldehyde is often used in automated tissue processors. The temperature used must be selected carefully, as thermal denaturation of tissue proteins will begin to occur at extreme temperatures. Generally 60°C is used with formaldehyde fixatives. The concentration of the fixative can affect the rate of fixation and the total penetration of fixative into the sample. Too high a concentration will lead to hardening of the tissue and the formation of excessive artifacts. Too low a concentration will allow exhaustion of the fixative before the process is complete.

1.1.4 Working Safely with Fixatives

Ethanol Methanol

Fixatives are among the most hazardous substances used in life science research. Work with these substances under the hood wearing gloves, lab coat and safety goggles.

Oxidizing Fixatives

Oxidizing agents such as potassium permanganate, potassium dichromate, and osmium tetroxide are powerful denaturants and are therefore of limited use. Osmium tetroxide is used most commonly in electron microscopy.

Formaldehyde is a suspect cancer hazard and a strong sensitizer. It is harmful if inhaled or absorbed through the skin. High exposures may be fatal. Formaldehyde can cause blindness if swallowed. Glutaraldehyde is corrosive, causing severe eye burns as well as severe irritation to the skin and respiratory tract. Additionally, glutaraldehyde can cause an allergic reaction.

Potassium permanganate

Potassium dichromate

Osmium tetroxide

Mercuric chloride may be fatal if swallowed. It is a birth defect hazard. Mercuric chloride is harmful if inhaled or absorbed through the skin. Mercuric chloride causes severe irritation to the eyes, skin, and respiratory tract.

Picric Acid Fixatives

Foremost among the picrate based fixatives is Bouin’s solution. Its mechanism of action is unknown. Like the mercurials, they give good nuclear detail but with less brittleness of tissue.

Safe Disposal of Aldehyde Waste Picric acid

1.1.3 Factors Affecting Fixation Fixation protocols are usually straightforward. The tissue is cut to dimensions suited to the rate of penetration of the particular fixative and placed in the fixative solution. The number of factors affecting the fixation process includes buffering, penetration, volume, temperature and concentration. In fixation pH is critical. This is especially the case with formaldehyde, where acidity favors the formation of formalin-heme pigment deposits in tissue and may lead to protein denaturation and structural deformations. Sources of acidity include hypoxia of tissues and oxidation of formaldehyde stocks to formic acid. To prevent extremes of acidity or basicity, fixatives are generally buffered to a pH between 6-8. Common buffers include phosphate, bicarbonate, cacodylate, and veronal. Commercial formaldehyde fixatives are often buffered with phosphate at a pH of 7. The diffusibility of each fixative will determine its rate of penetration 110

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Potassium permanganate, potassium dichromate and osmium tetroxide are strong oxidizers and can cause severe burns to any area of contact, possibly fatal if swallowed or inhaled. Furthermore, potassium dichromate is a known carcinogen.

National Diagnostics’ Neutralin converts aldehyde waste into a noncorrosive, nontoxic polymer and water (nonhazardous waste as defined in Title 40 Code of Federal Regulations (United States)) 40 CFR 261.24(a). Neutralin reduces disposal costs and contributes to a safer work environment.

Histology Applications - Fundamental Histological Technique

1.2 Decalcification The removal of calcium deposits is essential for good embedding procedure. Decalcification is usually carried out between the fixation and processing steps (Section 1.3). Bone must obviously be processed in this way, but other tissues may also contain calcified areas. A variety of agents or techniques have been developed to decalcify tissues, each with advantages and disadvantages. Immersion in solutions containing mineral acids, organic acids, or EDTA are the predominant methods used. Electrolysis has also been tried. Strong mineral acids such as nitric and hydrochloric acids are used with dense cortical bone because they will remove large quantities of calcium at a rapid rate. As might be expected, these strong acids also damage cellular morphology. Mineral acid decalcifiers are not recommended for delicate tissues such as bone marrow. Because they are not as aggressive, organic acids such as acetic and formic acid are better suited to bone marrow and other soft tissues. Organic acids act more slowly than mineral acids, and will require extended treatments to decalcify cortical bone. Formic acid in a 10% concentration is the best all-around decalcifier. Some commercial solutions combine formic acid with formalin to fix and decalcify tissues at the same time.

Calci-Clear HS-104 Decalcification utilizing chelating and sequestering agents to efficiently remove solubilized calcium.

Calci-Clear Rapid HS-105 Very rapid, high quality bone and tissue decalcification. Tissue distortion and structure loss minimized. (pg. 98)

(pg. 98)

1.3 Processing Fixed Tissue Once fixed, the tissue must be treated to allow the cutting of the thin sections required for viewing under the microscope. The procedures designed to prepare the tissue for sectioning are collectively known as Tissue Processing. First, the sample is dehydrated by immersion in a series of aqueous alcohol solutions gradually moving to pure alcohol. The tissue is then soaked in an appropriate solvent to remove the alcohol. Finally the tissue is embedded in paraffin wax, which enables the cutting of sections of between 3 and 10 microns thickness. The movement through the series of baths in Tissue Processing occurs either by hand or by means of an automated processor. Table 1.3a gives a typical tissue schedule for an automated processor. The optimum protocol for any particular sample and apparatus may vary.

Automated Tissue Processing Schedule Process Bath

18 hour cycle Time (hours)

24 hour cycle Time (hours)

70% Ethanol

1.5

80% Ethanol

1.5

95% Ethanol

1.5

Absolute Alcohol

1.5

Absolute Alcohol

1.5

Alcohol/Histo-Clear (v/v) 1.5

Histo-Clear

1.5

Histo-Clear

1.5

Histo-Clear

1.5

Paraffin

1.5

Paraffin

1.5

Paraffin

1.5

Table 1.3a

Histology

Products for Decalcification

Figure 1.3a An overview of the complete process of tissue preparation for viewing by light microscopy, from fixation to mounting.

Fundamentals

EDTA can remove calcium and is not as harsh as mineral or organic acids. The use of EDTA is limited by the fact that it penetrates tissue poorly and works slowly. It is also expensive in large amounts. Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. Electrolysis is slow and not suited for routine daily use.

1.3.1 Dehydration Dehydration is usually carried out by transferring the tissue through solutions of increasing alcohol concentration, until 100% alcohol is reached. Sometimes the first step is a mixture of Formalin and alcohol. Other dehydrants can be used, but have major disadvantages. Acetone, though fast, is a fire hazard, so it is safe only for small, hand-processed sets of tissues. Dioxane can be used without clearing, but has toxic fumes.

1.3.2 Clearing The step following dehydration is called “clearing” and consists of replacing the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The term “clearing” comes from the fact that the clearing agents often have the same refractive index as proteins. As a result, when the tissue is completely infiltrated with the clearing agent, it becomes translucent. This change in appearance is often used as an indication of the effectiveness or completeness of the clearing process. The most common clearing agent is xylene. Xylene is reasonably cost effective and works well for short-term clearing of small tissue blocks. Long-term immersion of tissue in xylene results in tissue distortions. Toluene is better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene. However, toluene is more expensive than xylene and more toxic, so toluene is less commonly used. Chloroform has been used in some applications, but it is a severe health hazard, acts slowly and may lead to sectioning difNational Diagnostics Clearing Agents Histo-Clear HS-200 Recognized as the best orange oil clearing agent available. Outperforms xylene while increasing safety. (pg. 100)

Histo-Clear II HS-202 Histo-Clear II substitutes for xylene especially well in dry mounting applications. Low citrus odor. (pg. 101)

Histosol HS-100 High flash point solvent replacement for xylene. (pg101)

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Histology Applications - Fundamental Histological Technique ficulties. Methyl salicylate is safe and effective, though rarely used due to cost. Orange oil based clearing agents, such as National Diagnostics Histo-Clear, offer the best clearing action with the lowest hazard rating of all xylene alternatives. Histo-Clear is excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of protocol. Because orange oils can break down to produce compounds which will interfere with staining procedures, it is important to use a product, such as Histo-Clear, which has been rigorously purified and then stabilized. Most tissue processing is done using automated machines that carry out the steps automatically. Tissues coming off a tissue processor are in a plastic box ready for the embedding stage.

Histology

Fundamentals

1.3.3 Embedding For mechanical support during the sectioning process, tissue must be infiltrated with an embedding medium. The usual embedding media are paraffin for light microscopy and an epoxy resin for EM samples. Paraffins are available that differ in melting point and hardness. Some products contain added plasticizers to make the blocks easier to cut. In general, the higher the melting point, the harder the wax. Waxes which melt at 55-58°C generally produce good sectioning results. In paraffin embedding, the tissue is infiltrated with the paraffin and placed in a mold containing molten paraffin which is then allowed to cool. Often a vacuum is applied inside the tissue processor to assist penetration of the embedding agent. It is important to orient the tissue in the paraffin to facilitate sectioning along the desired axis.

1.4 Sectioning Once embedded, tissues are cut into thin sections ready to be placed on a slide. This is done with a microtome, an apparatus for feeding the blocks past an ultrasharp blade with micron level precision. Paraffin blocks can be sectioned with high-carbon steel blades. Plastic blocks (methacrylate, araldite, or epon) are sectioned with glass or diamond knives. A glass knife can section down to about 0.1 micron. Ultrathin sections for electron microscopy (below 100 nm) are best done with a diamond knife. Sectioning tissues is an art. The selection of knife material, blade shape, cutting speed, knife angle and other variables must be determined through experience with the type of tissue and the particular equipment. Sections cut under non-optimal conditions will show tearing, ripping, “venetian blinds”, holes, folding, etc. (Section 1.5). As sections are cut, they are floated on a warm water bath to smooth out any wrinkles. They are then picked up on a glass microscope slide. The glass slides are then heated in a warm oven for about 15 minutes to help the section adhere to the slide. This step may be bypassed to preserve characteristics such as antigenicity. In this case, adhesive-coated slides may be substituted to pick up the sections. Typical adhesives for this purpose include starch, albumen, resins and combinations thereof. The adhered sections are then ready for further processing.

Tissue block

Epoxy resins were introduced to provide the high strength, ultrathin, thermostable sections required by electron microscopy. Araldite, Epon and others are available from a number of sources. Each has a different profile of strength, permeation rate, convenience, etc. The user is referred to the suppliers of these materials for technical information. Plastics require special reagents for dehydration and clearing that are expensive, limiting their use in light microscopy. The processing is usually done by hand and a special microtome is required for sectioning these blocks. Typically the technique is used for small biopsies, such as bone marrow or liver.

Alternative Embedding Media Compound

Material

Methyl methacrylate

Acrylic

Extremely hard. Very good for embedding bone without decalcification.

Glycol methacrylate

Acrylic

Very easy to work with.

Araldite

Epoxy

Similar to methacrylates, but requires a more complex embedding process.

Epon

Epoxy

Routinely used for electron microscopy where very thin sections are required.

Figure 1.4a Rotary microtome for light microscopy.

Characteristics

Table 1.3.3a

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Microtome knife

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1.4.1 Frozen Sections Sometimes in medical diagnosis it is necessary to perform a rapid analysis of a sample. This is facilitated by performing a frozen section. The piece(s) of tissue to be studied are snap frozen in a cold liquid or a cold environment (-20° to -70° Celsius). Freezing makes the tissue solid enough to section with a microtome. Frozen sections are performed with an instrument called a cryostat, a refrigerated box containing a microtome. The temperature inside the cryostat is about -20° to -30° Celsius. The tissue sections are cut and picked up on a glass slide. The sections are then ready for staining.

Histology Applications - Fundamental Histological Technique

1.5 Artifacts in Histologic Sections

1.6 Staining

Artifacts that appear in stained slides may result from a number of causes including improper fixation, the type of fixative, poor dehydration, improper reagents, or poor microtome sectioning.

Histological staining involves the use of dyes to highlight specific intra- or extracellular elements within tissue. A vast array of dyes and associated staining protocols exist in use. Each dye is targeted toward different cellular structures. The response to a given protocol can vary among samples. Many protocols are up to 100 years old, and were developed using partially characterized textile dyes. As a result, the detailed mechanism underlying many popular staining techniques is unclear.

The presence of a fine black precipitate on the slides, often with no relationship to the tissue (i.e., the precipitate appears adjacent to tissues or within interstices or vessels) suggests the formation of formalin-heme pigment. The identification of this problem can be confirmed by polarized light microscopy. The pigment is birefringent in polarized light and will appear as numerous bright white motes on the slide. It forms when the formalin buffer is exhausted and the tissue becomes acidic, which promotes the formation of a complex of heme and formalin. Formalin-heme pigment is most often seen in tissues containing large amounts of blood or heme proteins, or in autopsy tissues. Tissues such as spleen and lymph node are particularly prone to this artifact. Making thin sections and using enough neutral-buffered formalin (10 to 1 ratio of fixative to tissue) will help. If the fixative solution in which the tissues are sitting is extremely murky brown to red, place the tissues in new fixative.

Though alcohols such as ethanol make excellent fixatives for cytologic smears, they tend to make tissue sections brittle, resulting in microtome sectioning artifacts with chattering and a “venetian blind” appearance. Bubbles under the coverslip may form when the mounting medium is too thin, and, as it dries, air is pulled in under the coverslip. Contamination of clearing agents or coverslipping media may also produce a bubbled appearance under the microscope. It is important to confirm that a clearing agent is compatible with the mounting medium to be used because some solvent may be carried over to the mounting stage.

Formalin-heme pigment

Fold

Nick in cutting edge

Eosin B

Eosin B (excited)

Figure 1.6.1a The colors of the visible spectrum are represented above as three complementary pairs. The absorption of yellow light by the dye eosin produces a complementary purple color.

Histological Stains from National Diagnostics Harris’ Hematoxylin HS-400 For vibrant, reproducible H&E staining. Longest shelf-life of any Harris’ Hematoxylin on the market. (pg. 106)

Eosin Solution HS-402 1% stock solution. Ready for use in the protocol of your choice. (pg.106)

Alcian Blue HS-504 Histological stain used to differentiate acid and neutral mucopolysaccharides and as a differential stain for amyloid tissue. (pg. 107)

Fast Green FCF HS-516 Can be used as a protein stain, or as a substitute for Light Green SF yellowish in Masson’s Trichrome. (pg.107)

Amido Black 10B HS-601 Very sensitive stain for proteins found in blood. (pg.107)

Methylene Blue HS-525 Sensitive stain specific for RNA and DNA.

Basic Fuchsin HS-518 Used with periodic acid in the PAS stain for carbohydrates. (pg.107)

Methyl Green HS-606 Metachromatic amyloid staining, used in conjunction with Pyronin to differentiate RNA and DNA. (pg.107)

Biebrich Scarlet HS-506 Can be used as a plasma stain in Masson’s Trichrome. (pg.107)

Histology

Tissues that are insufficiently dehydrated prior to clearing and infiltration with paraffin wax will be hard to section on the microtome, leading to tearing and holes in the sections. Tissue processor cycles should allow sufficient time for dehydration, and the final ethanol dehydrant solution should be at 100% concentration, which can be difficult to achieve in humid climates. Covering or sealing the solutions from ambient air will help. Air conditioning (with refrigerants, not with evaporative coolers) will also reduce humidity in the laboratory. As a clearing agent, toluene is more forgiving of poorly dehydrated tissues, but it is more expensive and presents more of a health hazard than most other clearing agents.

The human eye responds to wavelengths of light between 400 and 700 nanometers (the visible spectrum). The presence of all wavelengths in this spectrum is perceived as white light. The presence of one wavelength alone is seen as a color: Blue for 450 nm light, Red for 600 nm light, etc. Furthermore, if one color (wavelength) is removed from the full visible spectrum, the light is perceived as having the “complementary color”. For example, materials which absorb at 450 nm (blue light) will appear carmine. In general, dyes appear colored because they absorb a particular wavelength in the visible region. The eye senses the reflected light as the complementary color.

Fundamentals

The presence of large irregular clumps of black precipitate on slides of tissues fixed in a mercurial fixative such as B-5 suggests that the tissues were not “dezenkerized” prior to staining. These black precipitates will also appear white with polarized light microscopy.

1.6.1 The Chemistry of Dyes

Bromophenol Blue HS-603 Dye for histones in alkaline and neutral buffer systems. (pg. 107)

(pg.107)

Pyronin Y HS-607 Used in conjunction with Methyl Green to differentiate RNA and DNA. Alternative marker dye for RNA electrophoresis. (pg.107)

Figure 1.5a Artifacts often found in histological sections. A) Formalin-heme pigment B) Fold which occurred during attachment to slide C) Line across section produced by a nick in the cutting edge of the microtome knife.

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Histology

Fundamentals

1.6.2 Why dyes produce color Absorption of light energy occurs when a compound has an electron which can be promoted by a “quantum permitted” mechanism to a higher energy level. The energy difference between the ground state and the excited state determines the wavelength of light absorbed. The energy absorbed can be re-emitted at a longer wavelength (fluorescence), or dissipated as heat (simple absorbance) (Figure 1.6.2b). All dyes possess a chromophore, an aryl ring system with one or more delocalized electrons. (Figure 1.6.2a). These electrons can be promoted to excited states by visible light. The absorption wavelength of a given ring system can be modified by the addition of non-aryl substituents (color modifiers). For example, the successive addition of methyl groups to the red dye Pararosaniline produces a series of dyes with progressively longer absorbance wavelengths: Methyl violet (4 methyl groups), Crystal Violet (6 methyl groups), and Methyl Green (7 methyl groups).

Basic Fuchsin

Methylene Blue

Figure 1.6.2a The molecular structures of dyes contain conjugated aromatic rings.

Figure 1.6.2b

Simple absorption vs. fluorescence.

1.6.3 The Chemistry of Staining Staining procedures provide conditions which promote the binding of a given dye to specific cellular organelles or extracellular features. The utility of a staining procedure lies in its ability to bind dye only to selected structures, highlighting these structures in contrast with the rest of the section. To accomplish this, each procedure makes use of a subset of possible interactions between the dye and the cellular components. The major classes of interaction (bonds) are ionic, covalent, and hydrophobic. Ionic bonding results from the attraction between positive and negative charges. In solution, acidic groups (carboxylic or sulfonic acids, etc.) will lose a proton and become negatively charged (anionic). Basic groups (generally amines) will accept a proton to become positively charged cations. The pH of the solution determines the extent to which any chemical group is protonated or deprotonated, and a dye or biological molecule may have many such groups on its surface. Thus, altering the pH of a staining solution will alter the charges on the dye and the tissue molecules, and therefore alter the staining pattern. Ionic bonds are the predominant mode of interaction between tissues and dyes. Covalent bonding occurs between uncharged atoms that require the gain or loss of electrons to reach a stable configuration. In the usual scenario, the atoms involved donate electrons to a shared orbital. The atoms then share the electrons involved, and are bonded by the resulting orbital. Coordinate bonds are a subclass of covalent bonds in which one of the atoms donates all the electrons (two of them) which are then shared by both of the atoms participating in the bond. Except with mordants, covalent bonds are of little importance in staining.

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Many dyes have a poor affinity for tissue when used alone. Various compounds, most often metal salts, have been found to enhance the staining of these dyes. These enhancing compounds are called mordants. The mechanism of action of the mordants is not clear, but it presumably involves coordination bonding between the metal and the dye, and then further coordination between this complex and the tissue.

1.6.4 Staining Procedures Most dyes used to visualize the membranes and organelles of the cell are water soluble. The embedded wax must therefore be removed prior to staining. This is done by effectively reversing the tissue processing schedule (Figure 1.3a).

Red Color Produced by Fluorescence

Red Color Produced by Absorption

The presence of nonpolar molecules in an aqueous environment forces water molecules to assume a highly ordered arrangement, which is entropically disfavored. A large sphere of nonpolar molecules presents less surface area to the water than many dispersed molecules, so nonpolar materials tend to aggregate into their own phase. This sort of behavior, where molecules partition out of an aqueous solution, is called hydrophobicity, and the tendency of hydrophobic molecules to self associate is called the hydrophobic interaction. This phenomenon is utilized in staining lipids, which are hydrophobic. Hydrophobic stains will tend to dissolve into lipid rich regions of the section, highlighting them for analysis.

There are literally thousands of staining protocols and procedures in use. As an example, one of the most common stains, the Hematoxylin-Eosin stain, is presented below. For a detailed list of stain procedures we recommend that you visit the University of Bristol web site: www.bristol. ac.uk/vetscience/services/pathology/

Hematoxylin and Eosin Stain:

Hematoxylin is a natural compound extracted from a species of tree found in Mexico and the West Indies. The extracted compound is then oxidized to produce hematein, which is the active staining component of the hematoxylin stain. Hematoxylin stains must therefore be “ripened” by oxidation before they can be used. Hematoxylin staining requires the use of a mordant (most commonly aluminum salts) and stains the nuclear components of cells a dark blue. Hematoxylin is used in combination with eosin because eosin stains the cytoplasmic organelles varying shades of pink, red or orange. The combination of the two stains provides a broad range of morphological information about the section.

Hematoxylin

Figure 1.6.4a The H & E (Hematoxylin and Eosin) stained section above shows dark blue, hematoxylin stained nuclei against pink, eosin stained cytoplasm. Thanks to the University of Bristol Department of Pathology and Microbiology.

Hematein

Eosin Y

Histology Applications - Fundamental Histological Technique

Protocol 1.6.4a H & E Stain with Ehrlich’s Hematoxylin FORMULATE HEMATOXYLIN AND EOSIN SOLUTIONS

Protocol 1.7a Mounting Slides with Histo-Clear and Histomount 1. Drain excess Histo-Clear from the slide by standing on end on a paper towel. Wipe excess Histo-Clear from the back of the slide.

Ehrlich’s Hematoxylin: 2g hematoxylin 100ml ethanol 100ml glycerol 100ml deionized water 10ml glacial acetic acid 15g potassium alum

2. Place the slide on a level surface, and apply a drop of Histomount using the dispenser rod.

Dissolve the dye in the ethanol. Add all other components and allow to ripen for 2 months in direct sunlight, or ripen immediately with 100mg of Sodium Iodate. Note that chemical ripening will shorten the shelf life of the product considerably. Eosin: 1g Eosin Y 100ml di Water

3. Hold the cover slip at a 45° angle to the surface of the slide, and allow the bottom edge to touch the drop of Histomount. When the drop has spread along the edge of the slip, let go of the slip and allow the Histomount to spread slowly (20-30 seconds). 4. Excess mounting medium may be removed while wet with a tissue, or with a razor blade when dry. Histomount will dry sufficiently to be read in 30 minutes. Full drying may require up to 48 hours. Drying can be accelerated at 37°C.

STAINING PROCEDURE 1. Stain rehydrated sections in Hematoxylin solution for 20-40 minutes. 2. Wash in tap water for 1-5 minutes, until sections turn blue (“bluing”)

5. Stain in Eosin Solution 10 minutes.

Hydromount HS-106 A nonfluorescing aqueous mounting medium, Hydromount is effective for frozen sections, amyloid, and immnofluorescent staining procedures. (pg. 105)

Histology

4. Wash 1-5 minutes in tap water, until blue.

Histomount HS-103 Histomount is a pH neutral, UV stabilized synthetic mounting media. Refractive index is matched to glass cover slips and slides. Produces museum quality mounts. (pg.105)

Fundamentals

3. Differentiate sections in 70% ethanol containing 1% HCl, for 5 seconds. This removes excess dye, allowing nuclear details to emerge.

Mounting Media

Omnimount HS-110 Specifically designed to be compatible with HistoClear II, Omnimount combines outstanding optical characteristics with low fluorescence and exceptional durability. (pg. 105)

6. Wash 1-5 minutes in tap water. 7. Dehydrate, clear and mount. Note the use of tap water in the washing steps- tap water provides the alkalinity necessary for the “bluing” process.

1.7 Mounting To preserve and support a stained section for light microscopy, it is mounted on a clear glass slide, and covered with a thin glass coverslip. The slide and coverslip must be free of optical distortions, to avoid viewing artifacts. A mounting medium is used to adhere the coverslip to the slide. Aqueous based mounting media are available, which allow the mounting of tissues directly from the staining procedure. However, the water solubility of some stains allows them to bleed and/or fade in such mountants, necessitating the use of resinous mounting media. To use a nonaqueous mountant, the section must first be dehydrated (again!) and cleared. Any water carried over to the mounting stage will show up as bubbles or vacuole-like structures, as the water droplets aggregate and distort the tissue. It is important to note also that the clearing agent used must be compatible with the mounting medium, or the sections must be thoroughly dried prior to mounting.

Figure 1.7a : To mount a slide, (A) Apply a single drop of mounting medium upon tissue section. (B) Hold coverslip at 45o allowing the drop to spread along the edge of the slip. (C) Let go of slip and allow medium to spread slowly. USA: 1-800-526-3867 EUROPE: 441 482 646022

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Advanced Histological Techniques

2.1 IMMUNOHISTOCHEMISTRY

2.2 ELECTRON MICROSCOPY

Antibody Binding / Detection Systems

Fixation / Processing / Sectioning / Staining

Histology

Special Techniques

Immunohistochemistry and Electron Microscopy...Getting Down to Details

wentieth century advances in science significantly increased the repertoire of the histologist. The develop ment of the understanding of the processes of the immune system led to the practice of labeling specific cellular constituents (antigens) by means of antigen-antibody interactions. The resulting field of Immunohistochemistry enables the identification of specific substances that cannot be characterized by conventional staining. Electron microscopy is another example of how advances in pure science have led to improvements in histological

technique. Modern physics gave us an understanding of the particle-wave duality of electrons. This understanding along with the ability to focus electron beams with magnetic lenses led to the development of electron microscopy, which represented a thousand fold improvement over the resolution achievable with conventional optics.

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Histology Applications - Advanced Histological Technique

2.1 Immunohistochemistry Immunohistochemistry is the application of Antibody/Antigen interactions to provide information about biological systems. The body’s response to the introduction of a foreign agent, known as the immune response, results in the production of antibodies which bind the offending material. Antibodies bind tightly and specifically to an “epitope” (one specific structure) on an “antigen” (foreign molecule or structure). The definition of an antigen is “anything that can be bound by an antibody”. This can be an enormous range of substances from simple chemicals, sugars, and small peptides to complex protein complexes such as a virus capsid. Not all antigens directly elicit an antibody response. Some require a carrier to be effective. These generally smaller antigens are called haptens.

re le

gio

iab

Va r

gio

Light chains

The antibody systems used in Immunohistochemistry can be broadly divided into two types, direct and indirect. With the direct method the visualizing agent is attached directly to the antibody that will bind with the antigen. The direct method is technically and theoretically straightforward, and yields results sufficient for many studies. Its sensitivity is limited by the fact that only one antibody, and therefore one visualization agent, is bound to each antigen.

Figure 2.1.1a With direct immunohistochemistry, the labeled antibody directly binds to the antigen.

Histology

Antigen binding sites

2.1.1 Antibody Binding

Special Techniques

The structure of an antibody resembles a ‘Y’. The stem of the ‘Y’ is the “constant region”, which defines the animal and class of the antibody. Antibody classes are designated by a letter, and prefixed with Ig (for immunoglobulin), thus IgG, IgM and IgE are all classes of antibodies, and all human IgG molecules have the same constant region. The arms of the ‘Y’ contain the variable regions of the antibody, where the antigen binding sites are located. Each arm has a binding site, so each antibody can bind two antigens.

Immunohistochemistry is generally carried out in sectioned tissue, which allows the antibodies free access to the interior of the cells. Immunohistochemistry can also be carried out on cells either in free solution or bound to membranes, or on monolayers of cultured cells. Intracellular Immunohistochemistry requires that the antibody to the target antigen be able to penetrate the cell membrane and whatever cell wall may be present before it can attach to the antigen. This requires a number of steps not required for sectioned tissue. Primarily the cell membrane must be made permeable to the antibody, though at the same time the integrity of the cell contents and structures must be maintained. This is normally achieved through the use of a specialized buffer containing a detergent. Saponin is frequently used for this purpose.

Indirect immunohistochemistry utilizes a second antibody specific to the first (primary) antibody. This secondary antibody has the visualizing agent attached. This allows for signal amplification. Primary antibodies directly label the antigen. The secondary antibody binds to the constant region of each primary antibody, with many secondary antibodies binding to each primary. This creates a cascade effect, amplifying the signal.

Heavy chains

Figure 2.1a Antibody structure.

Antibodies can be generated by injecting animals with antigens, and then collecting serum after the immune response has taken place. If the antibodies are labeled with an easily detectable molecule (a fluorescent dye, an enzyme, etc.), they become powerful detection reagents for the antigen. This system has been exploited to generate exceptionally specific and sensitive “stains” which are used in histology, as well as other disciplines. Immunohistochemistry/cytology is the use of antibodies in light microscopy and EM. The basic process depends upon selecting an antibody sufficiently specific to bind an antigen in situ. The antibody / antigen conjugate is then identified using a variety of signal generating molecules triggered either by the antibody/antigen interaction or by secondary processes. The signal generators can be precipitating dyes, fluorescent molecules or electron dense (ultrastructural tag) materials for electron microscopy (EM). The first report of an immunohistochemistry technique was made in 1942 when Coons et al detected pneumococcal antigen using a fluorescently tagged antibody. The immunohistochemical techniques for EM were developed by Singer using ferritin (1959). This was quickly followed by the first use of an enzyme, horseradish peroxidase (still widely used) in 1966 by Graham et al. Since then, a variety of enzyme and heavy metal techniques have been developed, the most important of which are Colloidal Gold for EM (Faulk 1971), Immunoperoxidase assay (Nakane, 1966), peroxidase/anti-peroxidase PAP technique (Sternberger 1970) and the Avidin-Biotin Complex (ABC) technique (Hsu, 1981).

Figure 2.1.1b The signal can be greatly amplified with indirect immunohistochemistry, in which the visualizing agent (horseradish peroxidase) is attached to polyclonal secondary antibodies with polyclonal primary antibodies binding antigen.

2.1.2 Detection systems Light microscopy makes use of primarily two detection systems for immunohistochemistry - fluorescence and enzyme labeling, while electron microscopy relies on the deposition of electron dense materials at the site of antibody binding. Techniques for light microscopy are discussed below. EM is covered briefly in the next section.

Immunofluorescence

The conjugation of a fluorescent dye to the primary or secondary antibody allows its detection under ultraviolet light. Microscopes are available which allow a slide to be UV illuminated, producing brightly emitting areas where the antibody has bound. Typically, Texas Red, Rhodamine, Fluorescine, or Phycoerythrin are conjugated to the antibody. Texas Red and Rhodamine fluoresce in the red range, Fluorescine in the green and Phycoerythrin in the blue. This allows multiple label experiments, in which different antigens show up as different colors. While extremely sensitive, this technique suffers from the fact that the dyes fade with time. Long term storage of slides is seldom practical, as loss of signal destroys their utility. USA: 1-800-526-3867 EUROPE: 441 482 646022

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Enzyme-Antibody Conjugate Methods

Methods have been developed to detect some enzymes at extremely low levels. These enzymes have been used as labels to facilitate detection of various molecules. Horseradish peroxidase (HRP) is often conjugated to antibodies, producing a system capable of detecting antigens by peroxidase staining. Incubation of HRP in a solution containing hydrogen peroxide and diaminobenzidine (DAB) results in the reduction of DAB to an insoluble brown precipitate, visible under the light microscope. Alkaline phosphatase (AP) is also used in an analogous method, in which case a phosphorylated naphthol is used as the substrate. The dephosphorylated napthol reacts with an azo dye such as Fast Red TR, to give a colored reaction product.

Histology

Special Techniques

A level of signal amplification is provided by the peroxidase-antiperoxidase complex system (PAP). This technique uses large complexes which contain many detection agents bound together. The binding of one of these complexes to each secondary antibody amplifies the signal manyfold. Either of these techniques may be combined with the Avidin-Biotin system to provide a more robust and easily generalizable detection method. Biotin is a small molecule which is bound tightly and specifically by the protein avidin. This binding has been exploited by attaching biotin to the secondary antibody (biotinylation), and avidin to the detection enzyme. Some of the advantages of this approach are: 1)

Projection lens

Objective lens

Condenser lens

Specimen

Anode Cathode

TRANSMISSION ELECTRON MICROSCOPE (TEM) Condenser lens

Scanning coil Specimen

Anode

Amplifier

Viewing screen

Cathode

Electron detector

SCANNING ELECTRON MICROSCOPE (SEM) Figure 2.2a: Comparison of the imaging paths of the transmission and scanning electron microscopes.

Only a small molecule is attached to the antibody, which minimizes the chance of interfering with the antibody-antigen interaction.

VIII

2) The same secondary antibody may be used with many detection molecules, simply by attaching avidin to each. 3) Use of an avidin-biotin complex as a bridging reagent amplifies the signal, as many detection molecules will bind to each biotinylated antibody. This is the same amplification mechanism outlined for the PAP system above.

Image on viewing screen

III

VII

Figure 2.1.2a The use of biotinylated antibodies and avidin attached detection enzymes increases sensitivity, flexibility and reliability.

2.2 Electron Microscopy The resolution of a microscope is limited by the wavelength of light passing through the sample. For visible microscopes using 400 nm light (blue light), the limit of resolution is one half the wavelength, or 200nm. This is some two to three orders of magnitude larger than many cellular structures. Electrons, like photons, have wavelike properties, but, unlike light, electrons can be accelerated to wavelengths well below 1nm. This has allowed the development of the Electron Microscope (EM), with resolutions down to below 1nm. Although electron wavelengths would theoretically allow resolution down to below 0.01nm, in practice, mechanical limitations on the construction of the apparatus have prevented this limit from being approached. Two types of electron microscopes are in wide use: the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The TEM operates on the same principles as a light microscope. A beam of electrons, accelerated from a tungsten filament, is focused on a sample, and the transmitted electrons are focused into an image. “Electron dense” areas of the sample (often made dense by staining techniques) scatter electrons, leading to dark areas in the image. The image itself, being made up of electrons, is invisible to the eye, so it is visualized by projection onto a fluorescent screen, which emits light when it is struck by electrons. For permanent records, photographic film, which is exposed by electrons, is used in place of the screen. 118

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Figure 2.2b : Of the organelles made clearly visible by the transmission electron micrograph of the hepatic cell above (rat), only the nucleus could have been resolved with a light microscope. Organelles: (I) Nucleus, (II) Endoplasmic reticulum, (III) Mitochondria, (IV) Golgi apparatus, (V) Bile canaliculus, (VI) Plasma membrane, (VII) Desmosome, (VIII) Secretory granule.

The SEM is used to visualize surface details of the sample. The image develops by means of the scattering of electrons by the surface of the sample when the beam hits it. A narrowly focused beam (10nm diameter) is “scanned” across the sample surface, and the secondary electrons which are reflected from the surface are amplified and used to determine characteristics of the sample surface at the probe position. The preparation of samples for transmission electron microscopy parallels tissue preparation for visible microscopy. Tissue samples must be fixed, processed, embedded, sectioned and mounted before viewing.

Histology Applications - Advanced Histological Technique The sections produced must be thinner and stronger than those for light microscopy, and the level of detail observable mandates that the tissue structure be exceptionally well preserved. Specialized fixing and processing techniques have been refined to meet these requirements. Furthermore, staining techniques have been developed to produce electron dense zones instead of colored or fluorescent areas. (Techniques of sample preparation for scanning electron microscopy are not covered here. For a good overview of the process, the reader is referred to: Robinson, G & Gray T. (1990) Electron Microscopy 3: Specialized Techniques, in Bancroft, J. & Stevens A. (ed.) Theory and Practice of Histological Techniques, 3rd edn. London. Churchill.)

2.2.1 Fixation

2.2.4 Staining Although secondary fixation in Osmium Tetroxide provides some areas of electron density, this is usually not sufficient to provide high contrast, high definition images. A number of staining techniques are available to enhance the contrast of areas of interest. These fall into two major categories. Positive stains deposit electron dense material on the area of interest, so that it stands out as a dark area on a light background. Negative stains penetrate and darken the interstices between areas of interest, which then appear light on a dark background.

Positive Stains

Histology

Osmium tetroxide fixes tissue by cross linking lipids. It reacts with unsaturated lipids and possibly with some proteins. Most proteins, however, are not well fixed by Osmium, and will leach out of the sample during processing. Osmium tetroxide may be used as a post- or secondary- fixative, after an aldehyde is used. This will stabilize membrane structures and also adds electron dense material to the section, enhancing contrast.

The ultrathin sections required in TEM are cut with knives of glass, diamond or sapphire. These materials produce extremely hard, ultrasharp edges, but they are brittle and subject to damage. Glass knives are produced as needed by fracturing. Sapphire knives and diamond knives may be purchased. The high cost of diamond knives makes resharpening economically feasible. Sections are cut into a trough mounted on the back of the blade, which is filled with water or 10-20% ethanol to “float out” the sections as they are cut. The refractive index of the sections will depend on their thickness, and this gives rise to apparent colors which can be used as a guide as to which sections are of usable thickness. Gold, silver and grey sections (ca 110, 75 and <60 nm respectively) are all usable for most purposes. Ultramicrotomy is challenging, and usually must be learned from an expert.

Special Techniques

The most popular fixatives for TEM work are the aldehydes and osmium tetroxide. Aldehyde based fixatives react with amines and other nucleophiles in the tissue, most notably lysine and arginine, generating cross-linked proteins. The cross linking action of these fixatives stabilizes the cytosol, preserving cellular structures. Aldehydes do not react with most lipids, so membrane components may be leached during fixing and processing. Glutaraldehyde has been used extensively. It is a protein cross linker, penetrates small tissue blocks acceptably (2-3mm penetration in 10 hours), and gives excellent fine structure preservation. Its main drawback is that it is a potent allergen, hazardous at levels above 0.05mg/ m3 (this is a mandated limit in some EU countries). Formaldehyde has been used for some purposes, but preservation of fine structure is not as good. One major benefit of formaldehyde fixatives is that formaldehyde is not as strongly denaturing to proteins as glutaraldehyde. Formaldehyde is therefore popular in immunohistochemistry. Mirsky’s Fixative from National Diagnostics is similar in action to glutaraldehyde, but Mirsky’s Fixative is not allergenic and poses a reduced health hazard. Mirsky’s Fixative is a good cross-linker and gives excellent fine-structure preservation. Its speed of action can be greatly increased by the use of microwave enhancement (Protocol 1.1.1a).

2.2.3 Sectioning

Uranyl acetate is used as a positive stain for EM. Uranyl ions react strongly with phosphate and amino groups, staining DNA and some proteins. Organelles composed of membranes are not stained well. Note that the starting material is radioactive. Lead citrate may also be employed as a positive stain. Reynolds lead citrate stain binds lead ions to negative ions, producing a general increase in contrast. Lead is a cumulative toxin, so skin contact must be avoided.

Negative Stains

Negative staining is most often used to highlight surface features on individual particles, such as virions, bacteria, or cell fragments. Table 2.2.4a lists a number of compounds used as negative stains. Negative Staining Agents for EM

Products for Fixation for TEM Mirsky’s Fixative HS-102 (Concentrate and Buffer) Excellent for immunohistochemistry with enhanced enzyme and antibody activity.

Mirsky’s Fixative HS-101 (Ready-to-Use) Convenient, no-mixing formula. 30 day shelflife. (pg. 103)

(pg. 103)

Neutralin HS-108 Improves safety by converting hazardous aldehyde waste into a nonhazardous polymer and water. (pg.104)

Stain

Formula

Solubility Density (g/100ml H2O) (g/cc)

Anionic Stains Ammonium Molybdate Sodium Phosphotungstate Sodium Tungstate

(NH4)6Mo7O24•4H2O

Na3PO4•12WO3 Na2WO4

2.5 3.8

4.2

Cationic Stains AgNO3

220

4.4

CdI2

5.73

Uranium Nitrate

UO2(NO3)2•H2O

150

3.7

2.2.2 Processing

Uranyl Acetate

UO2(C2H3O2)2•2H2O

2.9

Uranyl Formate

UO2(CHO2)2•H2O

3.7

Sections for TEM must be less than 80 nm thick in order to allow at least 50% of the electron beam to penetrate the sample. This can only be accomplished by using resins for embedding (epoxy, acrylic or polyester) which requires a modification of the processing protocol. Graded alcohol baths (typically 20, 40, 70, 90 and 100%) are used for the dehydration, but in place of the clearing agents used in light microscopy, a “transitional fluid” is used. This fluid is miscible with both ethanol and the embedding resin, and is almost universally 1,2 epoxypropane (propylene oxide). If water soluble embedding resins are used, the sample may be dehydrated in graded baths of the embedding resin instead of alcohol, and a transitional fluid is not needed.

Table 2.2.4a

Silver Nitrate Cadmium Iodide

Immunostaining

Antibodies bound to colloidal gold particles are visible under the EM as dark spots. The gold particles can range from 1 to 20 nm, although 5-10 nm seems to be an optimum range. Embedded or frozen sections can be probed in this way.

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Histology Applications - Useful Information

Useful Information for the Histology Laboratory

Histology

Useful Information

0.2M Acetate Buffer Compositions pH

Solution A (ml)

3.8

44.0

Solution B Xylene Cyanole (ml) (nucleotides)

Solution A (ml)

6.0

43.8

Solution B Xylene Cyanole (ml) (nucleotides)

6.2

4.0

41.0

9.0

6.2

40.7

9.3

4.2

36.8

13.2

6.4

36.7

13.3

4.4

30.5

19.5

6.6

31.2

18.8

4.6

25.5

24.5

6.8

25.5

24.5

4.8

20.0

30.0

7.0

19.5

30.5

5.0

14.8

35.2

7.2

14.0

36.0

5.2

10.5

39.5

7.4

9.5

40.5

5.4

8.8

41.2

7.6

6.5

43.5

7.8

4.2

45.8

Acetate buffer compositions: Solution A is 0.2M acetic acid (Dilute 1.2ml glacial acetic acid to 100ml with water). Solution B is 0.2M sodium acetate (dissolve 1.64g anhydrous sodium acetate in 100ml of water).

0.1M Citrate Buffer Compositions Solution B Xylene Cyanole (ml) (nucleotides)

Solution A (ml)

3.8

15.0

35.0

4.0

17.0

33.0

4.2

18.5

31.5

4.4

22.0

28.0

4.6

24.5

25.5

4.8

27.0

23.0

5.0

29.5

20.5

5.2

32.0

18.0

5.4

34.0

16.0

5.6

36.3

13.7

Citrate buffer compositions: Solution A is 0.1M sodium citrate (Dissolve 2.94g of sodium citrate dihydrate (mw294), in 100ml of water). Solution B is 0.1M citric acid (dissolve 2.1g citric acid•H2O in 100ml of water). Adapted from Bancroft, J.D. and Stevens, A. (eds) (1990)Theory and Practice of Histological Techniques. Churchill Livingstone, Edinburgh.

120

0.2M Phosphate Buffer Compositions

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Phosphate buffer compositions: Solution A is 0.2M monobasic sodium phosphate (Dissolve 3.12g NaH2PO4•2H2O in 100ml of water). Solution B is 0.2M dibasic sodium phosphate (dissolve 2.83g Na2HPO4 in 100ml of water).

Neutral buffered formaldehyde

General purpose fixative. Buffering minimizes formalin pigment formation. Formalin 100ml Distilled water 900ml Monobasic sodium phosphate 4g Dibasic sodium phosphate 6.5g

Formal Calcium

Preserves lipids better than standard formalin fixatives. Formalin 100ml Distilled water 900ml 10% aqueous calcium chloride 100ml

Scott’s Tapwater

Use when available tap water is too acidic for bluing of Hematoxylin-Eosin stains Potassium bicarbonate 2g Magnesium sulfate 20g Dissolve in 1 liter of distilled water.

Histology Applications - Suggested Reading

Suggested Reading

Fixation Aldehyde Based Fixatives

Staining

Enzyme-Antibody Conjugate Methods

Graham, R.C. and Karnovsky, M.J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. Journal of Histochemistry 14 291. Sternberger, L.A., Hardy, P.H. Jr, Culculis, J.J. and Meyer, H.G. (1970) The unlabeled enzyme method of immunohistochemistry preparation of antigen-antibody complex (peroxidase-anti-peroxidase) in identification of spirochaetes. Journal of Histochemistry and Cytochemistry 18 315. Nakane, P.K. and Pierce, G.B., (1966) Enzyme labeled antibodies: preparation and application for the localization of antigens. Journal of Histochemistry and Cytochemistry 14 929. Faulk, W.P. and Taylor, G.M. (1971) Immunocytochemistry 8 1081. Coons, A.H. et al (1942) The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J. Immunol. 45 159. Hsu, S.M., Raine, L. and Fanger, H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. Journal of Histochemistry and Cytochemistry 29 577-580.

Electron Microscopy

Meek, G.A. (1970) Practical Electron Microscopy for Biologists Wiley-Interscience, NY. Weaklye, B.S. (1974) A Beginners Handbook in Biological Electron Microscopy Churchill-Livingstone, Edinburgh, UK. Bullock, G.R. (1983) The current status of fixation for electron microscopy: a review. Journal of Microscopy, 133 1-15. Hayat, M.A. (1970) Principles and Techniques of Electron Microscopy: Biological Applications. 1 Van Nostrand-Reinhold, NY. Immunostaining Singer, S.J. (1959) Preparation of an electron dense antibody conjugate. Nature London 183 1523.

Artifacts in Histologic Sections

Wallington (1979) Artefacts in tissue sections. Medical Laboratory Sciences 36 3. Herschberber, L.R. and Lillie, R.D. (1947) Physical properties of acid formalin hematin, or formalin pigment. Journal of Technical Methods and Bulletins of the International Association of Medical Museums. 27 162.

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121

Reactive Oxygen - Oxygen Radical Assays

Oxygen Radical Chemical Assays Diogenes

l l l l

Superoxide

Oxygen Assay

APPL IC ATIO N Excellent for: Luminometric detection of superoxide. Superoxide dismutase assay using an intrinsic superoxide generator. NADPH oxidase assays. Neutrophil activation assays.

Cellular Luminescence Enhancement System for Superoxide Detection Easy to Use - Specific for Superoxide From 100-Fold to 600-Fold Enhancement Requires Fewer Cells - Non Cytotoxic

National Diagnostics’ Diogenes Cellular Luminescence Enhancement System is a superoxide chemiluminescent enhancer that is non-denaturing to living cells. Superoxide radical (O2¯) is produced intracellularly as a consequence of aerobic metabolism and extracellularly by leukocytes in response to infection. The extent of “oxidative burst” produced by white blood cells (WBCs) when stimulated by f-met-leu-phe, phorbol esters, anti-Fc receptor antibodies or LPS is a partial indicator of the immunocompetence of the cells tested. Currently, the production of O2¯ by leukocytes is monitored by such cumbersome and indirect methods as measuring oxygen uptake in a Clark electrode (both in the presence and absence of cyanide) or measuring spectral changes caused by the reduction of cytochrome c. As a non-cytotoxic intermediate in the mechanism of photon production, Diogenes is ideally suited to the detection of cell-mediated superoxide production. The intensity of light produced by Diogenes in the presence of superoxide is directly proportional to the O2¯ concentration, but is much higher than that achieved by using luminol. Therefore, Diogenes is ideal for monitoring cellular immunocompetence, utilizing a luminometer to quantify the light output. Any stimulant that activates an oxidase to produce extracellular superoxide is usable with Diogenes. Such means can be physiological or mimetic of the physiologic pathway. Storage: The prepared Diogenes Complete Enhancer Solution can be stored at 4-8OC for up to 30 days. The shelf-life of the non-reconstituted reagents in the original packaging is one (1) year. Product Name Diogenes Kit

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Cat. No.

Size

CL-202 1 Kit

Reactive Oxygen - Oxygen Radical Assays

Hydrogen Peroxide Assay Kit

l l l

Sensitive, Quantitative Assay for Hydrogen Peroxide Detects as Little as 15ng/ml Easy-to-use Colorimetric System

Hydrogen peroxide is a part of the oxygen reduction pathway, produced by the two-electron reduction of molecular oxygen, or by the one electron reduction of superoxide anion radical. Hydrogen peroxide is a potent oxidant, and the levels of hydrogen peroxide must be accurately determined in order to fully characterize the oxidative state of the system under study.

APP L IC ATIO N Excellent for: Sensitive, quantitative detection of hydrogen peroxide.

National Diagnostics Hydrogen Peroxide Assay Kit is a rapid, sensitive and quantitative method for the determination of hydrogen peroxide in chemical or biological systems. The assay is based upon formation of a complex between Xylenol Orange and ferric iron, which is produced by the peroxide dependent oxidation of ferrous iron. This reaction is quantified colorimetrically, detecting as little as 15ng/ml of peroxide.

Oxygen Assay

Protocol:

Hydrogen Peroxide

National Diagnostics Hydrogen Peroxide Assay is the ideal complement to our well established Diogenes Superoxide detection system. Using these kits in tandem, researchers can now quantitatively determine the first two steps in the oxygen reduction pathway.

1. Reagent Preparation:

Prepare 1.8 ml of reagent per sample to run tests in duplicate.

To prepare 20ml: Combine 19.8ml of Component A with 0.2ml Component B.

The mixture is stable at room temperature for one day.

2. Assay procedure:

Mix 0.9 ml of Assay Reagent with up to 0.1 ml of sample.

Incubate at room temperature for at least 30 minutes to allow for complete color development.

Read absorbance at 560nm.

Product Name

Cat. No.

Size

Hydrogen Peroxide Assay Kit

CL-204

1 Kit - 100 Assays

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123

Reactive Oxygen Assays - Reactive Oxygen Assays

Reactive Oxygen Species

Superoxide

Oxygen is used by a great variety of organisms as a means for producing energy. The redox potential of the oxygen: water couple is 1.229 Volts, meaning that a relatively large amount of energy is released during the 4 electron reduction which converts O2 to H2O. The incorporation of this reaction into cellular metabolism was an enabling step on the evolutionary path to higher organisms. However, harnessing this new resource came at a cost: the intermediates produced during the reduction of molecular oxygen are all highly reactive, and pose risks to the cells that use this pathway. The intermediates of oxygen reduction are shown below:

Superoxide has been implicated in diseases ranging from alzheimers to diabetes. The wide range of pathologies associated with superoxide is the result of its ability to react with a variety of cellular targets, including enzyme active sites, nucleic acids and lipids. Cells protect themselves against superoxide damage by producing superoxide dismutase enzymes (SODs). These enzymes accept an electron from a superoxide molecule, reducing the active metal (copper/Zinc, iron or manganese) at the core of the enzyme, and releasing oxygen. The reduced enzyme then reacts with a second superoxide, reducing it to produce hydrogen peroxide, and regenerating the enzyme. The high reactivity of superoxide in aqueous solutions almost precludes its being added to a biological test system in a controlled manner. As a result, much of what is known of the involvement of superoxide in disease states comes from studies in which SOD has been inactivated, leaving the cells vulnerable to endogenous superoxide damage.

O2 .

O2 + e O2 .

Superoxide

2 H2O2

e +

Detection of Superoxide

H2O2

Hydrogen Peroxide _

. OH

Fundamentals

Oxygen Assay

The first reduction product of oxygen is the superoxide radical ( O2- ). Carrying an unpaired electron, superoxide is a potent oxidizing agent. It has been found to react with numerous cellular structures, such as iron-sulfur clusters, unsaturated lipids, and nucleic acids. Superoxide can also cause biological damage by acting as a reductant, as in the Fenton reaction described below. Reduction of superoxide yields hydrogen peroxide, H2O2 . Hydrogen peroxide is also a powerful oxidant, and is commonly used in first aid as a biocide to cleanse wounds, or in high concentrations as a bleaching agent. While superoxide carries a charge, and is thus unable to freely cross biological membranes, H2O2 is uncharged, and can diffuse across membranes as easily as water. Hydrogen peroxide is also more stable than superoxide, and can diffuse through a cell or tissue, causing damage at a distance from its point of origin. In the presence of catalytic amounts of iron, superoxide and hydrogen peroxide can react to produce hydroxyl radical (OH. ).

2) Fe II +

Fe III

H2O2

Fe II

Hydroxyl radical is one of the most potent oxidizing agents known. It is too reactive to diffuse far in a cellular environment rich in targets for oxidation. It is thought to cause damage in the vicinity of iron centers or other sites containing bound iron. Oxidation by hydroxyl radical results in the concommitant reduction of the radical to water:

H 2O

Where X is the molecule oxidized by hydroxyl radical.

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A number of methods have been developed to detect superoxide. They all employ what has been termed an “indicating scavenger;” that is, a molecule which reacts with superoxide, producing a detectable product. The most commonly used indicating scavengers are cytochrome c, lucigenin and luminol, each of which has its own advantages and pitfalls. Cytochrome c is reduced by reaction with superoxide, producing ferrocytochrome c, which has a detectable absorbance at 550 nm. This assay is relatively insensitive, and is subject to a number of interferences from other chemicals and from enzymes which reduce the cytochrome directly. Lucigenin is a di-acridinium compound, which emits light on reaction with superoxide. The reaction involves an initial reduction of the lucigenin to a radical. The lucigenin radical can then react with either oxygen—producing superoxide—or with superoxide in an addition reaction—leading to the decomposition of the lucigenin into two acridones, one of which is in an excited state, and decays to produce light. As with most chemiluminescent reactions, lucigenin is more sensitive than colorimetric methods such as cytochrome c reduction. The lucigenin assay has been criticized as a measure of superoxide because lucigenin itself can react to produce superoxide, and because in some cases lucigenin has been observed to stimulate superoxide production by intact cells. Luminol—unlike cytochrome c and lucigenin—is oxidized by superoxide. This leads into a complex series of reactions between luminol, luminol radicals, oxygen and superoxide, ultimately producing a luminol endoperoxide, which decomposes with the release of a photon. Again, because superoxide is involved as both initiator and intermediate in the reaction, objections have been raised to the use of luminol as a quantitative measure of superoxide production. No matter which assay is used it is important to include the proper controls in the experimental design in order to be certain that any signal detected is due to superoxide. The primary control experiment is to run the reaction in the presence of superoxide dismutase (SOD). If the signal is not abolished by SOD, it does not represent superoxide production. However, the converse is not always true: a signal which is quenched by SOD does not absolutely indicate that superoxide is being produced by the test system. Both luminol and lucigenin can generate superoxide as they react to produce light, and both chemicals can undergo a redox cycle, induced by cellular agents, which produce light by way of a superoxide intermediate. Thus, in special cases, SOD will inhibit a signal which did not originate with a superoxide molecule, and results must be interpreted with caution.

Reactive Oxygen Assays - Reactive Oxygen Assays

Hydrogen Peroxide Hydrogen peroxide—like superoxide—can react with a variety of targets in the cell, and has been associated with a number of diseases. Aerobic organisms express catalase and peroxidase enzymes to prevent damage by H2O2. Peroxidases—most of which contain a heme at their active site—undergo an enzymatic cycle in which a molecule of peroxide oxidizes the enzyme, releasing one of the peroxide oxygen atoms as water, and leaving the other complexed to the enzyme-bound iron. This configuration is known as Compound I. The second part of the cycle involves a transfer of electrons from a reducing substrate to the enzyme, releasing the bound oxygen as water, and regenerating the starting configuration of the active site. The net reaction is:

H2O2

2 AH

2 H2O +

where AH is the reducing substrate. Catalase is a specific sub-form of peroxidase, in which the reducing substrate is a second molecule of hydrogen peroxide. In this case the reaction is:

2 H2O2

2 H2O +

Perhaps the prototypical example of a peroxidase is horseradish peroxidase (HRP), which is frequently used as a label in immunochemistry. Coupled to an antibody, HRP can be sensitively detected by its catalytic oxidation of luminol in the presence of hydrogen peroxide. The oxidized luminol is metastable, rearranging to give off a photon of light.

A number of other assays have been developed to measure hydrogen peroxide levels. Many of these are based on the horseradish peroxidase mediated reaction between H2O2 and some indicating reactant. For example, in the presence of H2O2 , HRP will oxidize luminol, producing light. Under the proper conditions, the light output of this system can be made to be proportional to the concentration of peroxide present. This assay can be extremely sensitive, but it is also subject to many interferences, as HRP can react with a variety of different cellular substrates. Regardless of the assay used, two possible sources of error must be controlled for. The first is interference from compounds other than H2O2 which are redox active and can create a signal within the assay. such interferences can be detected by running the assay after treating the sample with catalase, which will specifically remove the H2O2 . Any signal which is lost to such treatment can be confidently assigned to H2O2 . The second source of error often encountered is the scavenging of peroxide by components of the sample either before or during the assay. Cells contain elaborate defenses against peroxide damage, and such systems often continue to work during sample preparation procedures. It is therefore important to prepare samples under conditions which minimize peroxide degradation, ie short post preparation storage time, prep and storage at or below 4OC. It is also possible to “spike” a sample with a known amount of peroxide during work-up. Comparing the amount of peroxide detected in spiked vs. unspiked samples will give an indication of the extent of peroxide degradation between sampling and assay.

Detection of Hydrogen Peroxide

Diogenes CL-202 Cellular luminescence enhancement system for superoxide detection. Easy to use. Specific for superoxide. (pg.122)

Hydrogen Peroxide Assay Kit CL-204 Sensitive, quantitative assay for hydrogen peroxide. Easy-to-use, colorimetric system. (pg.123)

Fundamentals

Products for Assay of Reactive Oxygen

Oxygen Assay

In at least one important way, measuring hydrogen peroxide is substantially easier than measuring superoxide. Superoxide is unstable in aqueous solution- its steady state concentration cannot be measured directly. As a result, superoxide “levels” must be determined indirectly, by measuring rates of production and removal, and then correlating the two, or by measuring damage to a superoxide target. Hydrogen peroxide, in contrast, is relatively stable, allowing direct measurement of it’s concentration in many cases. The classical methods for measuring hydrogen peroxide concentrations are through direct measurement of the absorbance at 330nm of the H2O2 molecule, or through reaction of the peroxide with ferrous iron, monitored via a subsequent reaction with the dye xylenol orange. A variety of other methods are also available. While H2O2 does not have an absorbance peak at 330nm, the absorbance at this wavelength correlates well enough with the concentration of H2O2 to allow its use as a quantitative assay, using an extinction coefficient of 43.6/Mcm (JBC 245 (9) (1970) pp2409-13). Of course, given the low extinction coefficient, this is not a very sensitive assay. In addition, while 330nm is a long enough wavelength to minimize interferences from DNA and aromatic amino acid residues, many cellular components will show a significant absorbance at this wavelength. However, for measuring high concentrations of H2O2 , and for calibrating standard solutions, this measurement can be very useful. A higher sensitivity, more selective assay is mediated by the oxidation of ferric iron to its ferrous state. The ferrous iron so produced will form a complex with the dye xylenol orange, resulting in a net increase in absorbance of the solution at 560nm. This assay is much more sensitive, being capable of detecting as little as 15ng H2O2 per ml. It is, however, subject to interferences from compounds which can oxidize ferric iron, and so requires the use of the appropriate controls as described below.

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125

Liquid Scintillation Products - Scintillation Cocktails

S c in t illati o n C o c k tails National Diagnostics is recognized as a world leader in the manufacture and supply of scintillation cocktails, combining manufacturing excellence with safe and environmentally sound practice. Our innovative approach to product development led directly to the introduction of the world’s first biodegradable scintillation cocktails.

N o n aqu e o u s S ampl e s

Monofl ow 1

p.138

nonaqueous sample

Ecoscint Flow

Fl o w L S C

p.136

the highest performing flow cocktail

Monofl ow 5

S af e r

A qu e o u s S ampl e s

p.137

moderate sample volume

Uniscint BD p.132 high salt

T r adi t i o n al

Vi a l LSC

Monoflow 2

p.138

all-around performance

Monoflow 3

p.138

high sample volume

Monoflow 4

A qu e o u s S ampl e s

high salt

T r adi t i o n al

No na q ueous Sa m p l es

Hy d r ofl uor

p.133

good sample volume “universal” solution

Monofluor p.134

LSC

LSC Cocktails

no gel phase highest sample volume

Liquiscint p.135 low cost

Soluscint XR p.141

Ecoscint O

S af e r

Sp ec i a l Applications

Safer

high salt

Ecoscint Ultra p.128

p.131

best all-around cocktail on the market

nonaqueous sample

Ecoscint XR p.129

T r a d i t i on a l

ultra-high sample hold

B e ta fl u o r

p.135

Ecoscint A p.130

nonaqueous sample

excellent, balanced performer

Ecoscint H p.130

Sa m p l e Oxi d a t i on

O xo s o l C 1 4

optimized for efficiency

Ecoscint ORIGINAL p.131 the first biodegradable scintillation solution

p.139

absorbs and counts 14CO2 produced by combustion

Filter/Tissue Solubilization

Carbamate-1 p.139 CO2 absorber

Oxosol 306 p.139 cocktail formulated to count samples trapped in Carbamate-1.

F ilt r on-X p.141 dissolves membrane filters

Biosol/Bioscint p. 140 biodegradable tissue solubilizer/ scintillator system

Ecoscint GL 132 for counting resins, silica and other fine particulates Solusol p.140 tissue solubilizer

Soluscint XR p.141 specially formulated cocktail for use with Solusol

126

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Uniscint BD p.132 high salt

p.138

Liquid Scintillation Products - Scintillation Cocktails

Choo sing a Sc i n t i l l a t i on Fl ui d for Y o u r Sa m p l e or A p p l i c a t i on S c i n t i l l at i o n F l u i ds 39 (p ) g. 13 5)

o fl o w (p on ow 1 g.1 o M fl o (pg 36) on w .1 2 38 o M fl o (pg ) on w .1 3 38 o M fl o (pg ) on w .1 4 38 o F i fl o (pg ) ltr w .1 o 5 38 B i n -X (pg ) os .13 ( o pg. 7 S o l / B 141) ) io lu s s So ol ( cin l u pg. t (p s c 14 g. 14 0) O i 0) xo nt s o XR (p C l g ar C 1 4 .141 b ) Be am (p t a a t e g.13 fl u 9 ( o r pg.1 ) M

tra ( X R pg.1

S a m p l es an d Applications

28 (p ) g. 12 9 ci (p ) g. Ec nt os H 130) ci (p g. Ec nt os O 130) ci (p U n g n i t O .131 sc RI ) G Ec i n t IN os A cin B D L (p g. t (p H y d GL g.13 131) 2) r o (pg M fl u .132 on o ) o r( L i fl u pg.1 qu o 3 i s r ( 3) E c c i n pg.1 o s t ( 34) p c M i n t g.13 on F l 5)

(green = biodegradable)

LSC

LSC Cocktails

Acidic samples Agarose gels Alkaline samples Alpha counting Alpha/beta discrimination Ammonium formate Ammonium phosphate Aqueous samples - high vol. Aqueous samples - low/med. vol. Biological samples Blood Brain Carbon dioxide Catecholamine assays Cellulose acetate membranes Cellulose nitrate membranes Cesium chloride gradients Density gradients Environmental samples Filter paper Flow counting - high efficiency Flow counting - high salt Flow counting - high sample hold Gaseous samples Gelling scintillation counting Glass fiber filters High sensitivity counting HPLC gradients Inositol phosphates Milk Organic samples - hydrophobic Oxidation counting Plasma Polar (hydrophilic) solvents Polyacrylamide gels Powders - suspended Radon RIA - supernatants Salt solutions - high Salt solutions - medium, low Serum Sodium hydroxide gradients Solids Solubilization Sucrose Tissue homogenates Tissue solubilization Urine Water

best performance

good performance USA: 1-800-526-3867 EUROPE: 441 482 646022

127

Liquid Scintillation Products - Biodegradable Scintillation Cocktails for Vial Counting

Biodegradable Scintillation Cocktails

Recognizing the need for enhanced safety and environmental friendliness in the laboratory, National Diagnostics was the first company to introduce biodegradable solvents for scintillation counting. The products in this section are formulated with low toxicity, nonflammable solvents. National Diagnostics biodegradable scintillation cocktails are optimized for the highest possible quality and performance while minimizing risk to the operator and the environment.

Ecoscint Ultra TM

l l l l l

For Environmental Sample Counting Ultra-High Water Capacity (1:1.2 Cocktail to Sample) Ultra-Low Background Unsurpassed Counting Efficiency Ideal for Low Temperature Environmental Counting

APPLICATIONS Excellent for: High or low volume aqueous samples, environmental samples, biological samples, acidic samples, alkaline samples, polar solvents, medium/low salt solutions, urine. Counting discrete samples....................................................... p.156

When National Diagnostics laboratories set out to create Ecoscint Ultra, our aim was to create the best cocktail available for environmental sample counting and urine bioassay. We are proud to announce that not only have we succeeded in this goal, but we have surpassed it, creating the best all-around performing cocktail on the market for both large and small aqueous samples.

Safer Cocktails

Ecoscint Ultra enables ultra-low level counts to be discriminated from background not only for large but also for small samples. With large samples, Ecoscint Ultra delivers up to 30% 3H counting efficiency at maximum sample hold with very low background levels. With small samples, Ecoscint Ultra yields 3 H efficiency greater than 60%. o

LSC

Ten millilters of Ecoscint Ultra holds 12ml H2O in a clear emulsion at 18 C, while 10ml of Ecoscint Ultra holds 10ml 0.5M NaCl at that temperature. Sample Hold Capacity (ml) in 10ml Ecoscint Ultra* 0

2.0

4.0

6.0

8.0

Distilled Water 0.5M NaCl 0.1M NaCl 0.1M NaH2PO4 0.1M Tris

12 mL 8 mL 10 mL 10 mL 10 mL 1X PBS 10 mL

* Ecoscint Ultra was designed both for low temperature (15OC), ultra-low background counting and room temperature counting. The data above reflects the intermediate temperature of 20OC. For salt or buffer solutions at 25OC, we recommend verifying emulsion clarity before exceeding 6ml sample in 10ml cocktail.

Product Name

Cat. No.

Size

Ecoscint Ultra LS-270 4 liter (1-3) 4 liter (4 +)

128

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Liquid Scintillation Products - Biodegradable Scintillation Cocktails for Vial Counting

Ecoscint XR

l l l l l

Biodegradable Ultra-High Sample Holding (1:1 Cocktail to Sample) Excellent Counting Efficiency Low Toxicity High Flash Point

APP L IC ATIO N S Excellent for: High volume aqueous samples, environmental samples, biological samples, acidic samples, alkaline samples, polar solvents, medium/ low salt solutions, sucrose, urine. Useful for: Low/medium volume aqueous samples. Counting discrete samples.... p.156

Ecoscint XR is an excellent universal scintillation fluid, able to count large aqueous samples, non-aqueous samples, and practically anything in between. 10ml of Ecoscint XR can hold up to 10ml of most common aqueous samples, easily accepting high pH, low pH, or high salt samples. Ecoscint XR mixes rapidly to produce a homogeneous, clear emulsion. Unlike many fluids, Ecoscint XR does not sacrifice performance to achieve robust sample hold. Ecoscint XR produces counting efficiencies of up to 55% 3H and 90% 14C with small volume samples. Sample Hold (Volume of Sample per 10ml Ecoscint XR) Water Sodium Hydroxide 0.1 N Sodium Chloride 0.5 M Sodium Chloride 1 M Sucrose 40% Ammonium Acetate 15 mM Ammonium Acetate 0.25 M Tris/HCl 0.05 M pH = 7 Tris/HCl 0.5 M pH = 7 Washing Buffer 1X PBS Ammonium Sulfate 0.1 M Methylene Chloride Acetonitrile Ammonium Fomate 1 M

Cat. No.

Size

Scin til lation Vials High density polyethylene (HDPE) scintillation vials, manufactured as a onepiece molding with no seams, preventing cracking, pinholes and leakage. These vials provide excellent UV light transmission for high counting efficiency.

LSC

Ecoscint XR LS-272 4 liter (1 - 3) 4 liter (4 +)

Safer Cocktails

Product Name

10 mL 8 mL 10 mL 5 mL 150 ÂľL 10 mL 10 mL >10 mL 10 mL 10 mL 10 mL All proportions All proportions 4 mL

S c i n ti l l a ti o n V i a l s 20ml Scintillation Vials with Screw Caps (1000/case) [pg 144]

SVC-20 1-10 cases 11+ cases

8ml Scintillation Vials SVC-08 with Push-on/Twist-off Caps (2000/case) [pg 144]

1-10 cases 11+ cases

6ml Scintillation Vials SVC-06 with Push-on/Twist-off Caps (1000/case) [pg 144]

1-10 cases 11+ cases

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129

Liquid Scintillation Products - Biodegradable Scintillation Cocktails for Vial Counting

Ecoscint A

APPLI C A TION S Excellent for: Agarose gels, aqueous samples, biological samples, environmental samples, glass fiber or paper filters, plasma, polyacrylamide gels, salt solutions, sucrose, solublized tissues.

Ecoscint A displays exceptional sample holding capability while still delivering high efficiency. Ecoscint A will easily accommodate up to 40% sample while maintaining a single liquid phase. Furthermore, Ecoscint A has exceptional resistance to photoluminescence and chemiluminescence. Ecoscint A is readily biodegradable, with a mean DOC elimination of >70% at 10 days. The low odor and low toxicity of Ecoscint A make it perfect for bench-top work. It is not necessary to use or store Ecoscint A under a fume hood. % Counting Efficiency (3H) of Typical Samples at Various Volumes (in 10ml Ecoscint A)

Useful for: Acidic samples, alkaline samples, catecholamine assays, high salt solutions, tissue homogenates.

Sample Volume Sample Distilled Water 0.15M NaCl 0.01M PBS 0.05M Tris-HCl 0.01N NaOH 10% Sucrose BSA (1mg/ml) Urine 8M Urea 0.1M HCl

Counting discrete samples....................................................... p.156

Figure 1 - Sample Holding Capacities of Ecoscint A (10ml)

Sample Volume (ml) 1.0

2.0

3.0

High Efficiency with High Sample Holding Biodegradable, Reduced Toxicity Solvent

4.0 4.5

Distilled Water 0.15M NaCl 0.01M PBS

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

56.2

51.8

50.4

47.6

45.8

42.8

42.5

39.5

37.8

35.6

56.2

51.4

49.9

47.7

46.8

42.5

42.1

40.3

39.5

56.2

51.6

49.6

47.7

45.0

43.6

42.0

40.5

38.1

56.2

52.0

50.3

48.6

45.1

43.4

42.7

40.8

39.8

37.7

56.2

53.0

52.6

49.3

47.3

46.3

41.8

40.6

39.2

34.9

56.2

53.6

50.3

45.7

43.2

42.1

40.7

56.2

51.5

48.9

46.7

45.1

44.4

42.6

40.7

39.5

36.9

56.2

50.9

43.6

39.7

37.1

56.2

51.8

48.5

56.2

50.2

47.6

45.2

44.2

43.3

39.0

38.7

36.7

35.3

0.05M Tris-HCl

Product Name

0.01N NaOH 10% Sucrose

Cat. No.

Size

Ecoscint A LS-273 4 liter (1-3) 4 liter (4+) 20 Liter

BSA (1mg/ml) Urine 8M Urea 0.1M HCl

1.0

2.0

3.0

4.0 4.5

Ecoscint H TM

Ultra High Efficiency Biodegradable Scintillation Fluid

LSC

Safer Cocktails

Ecoscint H is specifically designed to provide the maximum counting efficiency for aqueous sample volumes under 10% of the total liquid scintillation volume. Furthermore, Ecoscint H has exceptional resistance to photoluminescence and chemiluminescence, which is especially important when counting biological samples or acrylamide gel slices. % Counting Efficiency (3H) of Typical Samples at Various Volumes (in 10ml Ecoscint H)

APPLI C A TION S Excellent for: High sensitivity counting, low volume aqueous samples, filter paper, glass fiber filters. Useful for: Acidic samples, biological samples, environmental samples, inositol phosphates, plasma, polyacrylamide gels, salt solutions, serum, sucrose, urine, RIA supernatants. Counting discrete samples....................................................... p.156

Sample Volume (ml) Distilled Water 0.15M NaCl 0.01M PBS 0.05M Tris-HCl 0.01N NaOH 10% Sucrose BSA (1mg/ml) 0.1M HCl

Product Name

Cat. No.

Size

Ecoscint H LS-275 4 liter (1-3) 4 liter (4+) 20 Liter

130

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Liquid Scintillation Products - Biodegradable Scintillation Cocktails for Vial Counting

Ecoscint O TM

l l l l

APP L IC ATIO N S Excellent for: Organic or hydrophobic samples, carbon dioxide and oxidation counting, alpha particle counting, radon extraction. Counting discrete samples....................................................... p.156

Cocktail for Non-Aqueous Samples Readily Biodegradable Excellent Counting Efficiency Low Toxicity

Ecoscint O is intended for use with nonaqueous samples. Ecoscint O is readily biodegradable, and because of its high flash point and low toxicity, Ecoscint O represents a substantial improvement in safety over conventional cocktails. Additionally, Ecoscint O exhibits lower backgrounds and improved quench resistance compared to non-biodegradable solutions. Product Name

Cat. No.

Size

Ecoscint O LS-274 4 liter (1-3) 4 liter (4+) 20 Liter

Ecoscint original TM

APP L IC ATIO N S

Ecoscint has comparable efficiency and greater sample holding capacity than conventional scintillators, and maintains a single liquid phase up to saturation. Product Name

Cat. No.

Size

LSC

Useful for: Alkaline samples, biological samples, milk, plasma, high salt solutions, homogenized or solubilized tissues. Counting discrete samples....................................................... p.156

Ecoscint—the first ecologically friendly scintillation solution—is still available from National Diagnostics. Ecoscint offers the convenience of a complete and ready to use solution, for both aqueous and nonaqueous samples. Substantially less toxic than conventional scintillation fluids, Ecoscint is biodegradable and nonflammable.

Safer Cocktails

Excellent for: Aqueous samples, agarose gels, paper or glass filters.

The Original Ecologically Responsible Scintillation Fluid Readily Biodegradable

Ecoscint LS-271 4 liter (1-3) 4 liter (4+) 20 Liter

Storage, Packaging, and Disposal of Biodegradable Scintillation Fluids Storage: All Ecoscint products and Uniscint BD solutions are best stored tightly capped in a cool dry area. No deterioration has been observed on storage for five years at room temperature, or heating to 50˚C for one week or cooling to -30˚C for one week. Packaging: All Ecoscint and Uniscint BD solutions are supplied in unbreakable 4-liter and 20-liter fluorinated high density polyethylene containers. Shipping weight is 9lbs. (4kgs.) per 4-liter bottle and 40 lbs. (18kgs.) per 20-liter drum. Disposal: U.S. Federal Nuclear Regulatory Commission and Environmental Protection Agency regulations permit the disposal of Ecoscint products through conventional sanitary sewage systems. Liquid scintillator counting solutions of Ecoscint products containing Tritium and/or Carbon-14 in concentrations of 0.05 microcuries or less per gram may be disposed of without regard to radioactivity. However, a record of such disposal must be maintained (10 CFR Part 20 Section 20.306). It is important to note that local regulations may differ from and supercede federal regulations. In this case, local regulations concerning the disposal of Tritium and/or Carbon-14 must be followed. Ecoscint products are considered “organic matter” and may be disposed of accordingly.

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Liquid Scintillation Products - Biodegradable Scintillation Cocktails for Vial Counting

Uniscint BD TM

Biodegradable Cocktail for High Salt Samples Low Toxicity

l l

Uniscint BD is a biodegradable scintillation solution especially formulated to accommodate high salt and buffer samples while still delivering efficiency. Uniscint BD has a low viscosity and is non-gelling. It is suited for use in online HPLC flow detectors as well as in traditional vial counting.

APPL IC A TION S Excellent for: High salt solutions, urine, ammonium phosphate or CsCl2 gradients, acidic samples, flow counting.

Uniscint BD accommodates all concentrations of ammonium phosphate up to 2M, at a ratio or 3:1 scintillator to sample, making it the ideal choice for counting ammonium phosphate gradients from 0-2M. Other samples, such as 2M ammonium formate and water are also accommodated at a 3:1 ratio.

Useful for: High volume aqueous samples, biological samples, plasma, RIA supernatants, glass fiber filters, sucrose gradients.

% Counting Efficiency (3H) of Typical Samples at 3:1 Uniscint BD to Sample Ratio

Counting discrete samples....................................................... p.156 Flow scintillation counting........................................................ p.158

Sample 2M Ammonium Phosphate 2M Ammonium Formate Water

Product Name

Cat. No.

% Count ing Effi ciency 32 33 35

Size

Uniscint BD LS-276 4 liter (1-3) 4 liter (4+) 20 Liter

Ecoscint GL Safer Cocktails

LSC

l l l

For counting isotopes bound to silica or resins Forms stable gel phase with water or moderate salt Reliable counting without settling of particulates

Ecoscint GL is the liquid scintillation cocktail to use for counting resins, silica and other particulate samples, as it alleviates the worry of solid materials drifting to the bottom of the vial. Ecoscint GL forms a semisolid gel phase, which allows for sample material to be suspended for counting. Ecoscint GL will hold up to 2 ml of sample per 10 ml of cocktail in a liquid emulsion, and will form a gel at 2.5-3.5 ml of sample with water or moderate salt. Product Name

Cat. No.

Size

Ecoscint GL LS-262 4 liter (1-3)

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Liquid Scintillation Products - Traditional Scintillation Cocktails for Vial Counting

Traditional Scintillation Cocktails As one of the first companies to manufacture scintillation cocktails commercially, National Diagnostics has a long history of providing quality cocktails for high efficiency counting of all samples. The following pages contain scintillation fluids for all applications which are formulated using a nonbiodegradable solvent.

Hydrofluor

l l l

Hydrofluor is a high efficiency, ready-to-use liquid scintillation solution designed to count large volumes of aqueous radioactive samples. Hydrofluor contains LSC grade fluors and solvents for one-step sample preparation. Up to 5 ml of aqueous solutions (acids, bases or biological fluids) can be incorporated in 10 ml of Hydrofluor and counted with high efficiency. Additionally, Hydrofluor has been specially designed to offer extremely low backgrounds and minimize chemiluminescence.

APP L IC ATIO N S Excellent for: High volume aqueous samples, gel phase counting of suspended solids, biological samples, filter papers, salt solutions, tissue homogenates, density gradients with sucrose or CsCl2.

Counting discrete samples....................................................... p.156

Hydrofluor Efficiency (Tritium) at Various Sample Volumes of Distilled Water

Storage: Hydrofluor is best stored tightly capped in a cool dry area.

LSC

Traditional Cocktails

Hydrofluor may be used to prepare samples in either liquid or gel form. Sample volumes up to 15% Hydrofluor volume form clear mobile solutions. Sample volumes from 25-50% Hydrofluor volume form stable countable gels which may be used to suspend solids such as TLC plate scrapings. Sample volumes between 15-25% Hydrofluor volume form a two-phase system which is not recommended for counting. Increasing the sample or scintillator content of these systems shifts them into the countable phase range (see graph).

Useful for: Acidic samples, alkaline samples, catecholamine assays, glass fiber filters, environmental samples, milk.

% Tritium Counting Efficiency

For Large Volume Aqueous Samples High Counting Efficiency Ready-To-Use for All Sample Types

40 Clear Mobile Liquid

30 20

Product Name Two Phase System

Cat. No.

Size

Hydrofluor LS-111 4 liter (1-3) 4 liter (4 +) Gel

10 0

20 30 % Water in Hydrofluor

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Liquid Scintillation Products - Traditional Scintillation Cocktails for Vial Counting

Monofluor

l l l l

APPLIC A TION S Excellent for: Agarose gels, aqueous samples, biological samples, environmental samples, carbon dioxide, glass fiber or paper filters, low salt solutions, urine, sucrose gradients, tissue homogenates. Useful for: Alkaline samples, polyacrylamide gels.

Extremely High Sample Holding Capacity Clear Continuous Liquid Phase to 50% Sample No Gel Phase High Efficiency

Monofluor is a complete liquid scintillation solution designed to count extremely large sample volumes. A 5.0ml aqueous sample can be counted as a crystal clear mobile liquid in 10ml Monofluor. With Monofluor, the suspension remains homogeneous with increasing sample volumes. There are no two-phase regions or non-homogeneous gel suspensions. The efficiency of Monofluor is nearly linear throughout its counting range without the dropoff encountered in gel phase counting.

Counting discrete samples....................................................... p.156

Typical Efficiencies for 10ml Monofluor 50 45

% Tritium

40 35

Product Name

Sample Volume (ml)

Cat. No.

Size

LSC

Traditional Cocktails

Monofluor LS-191 4 liter (1-3) 4 liter (4+)

Improve Radi ation Safe ty Nuclean and Nuc-Wipes Nuclean and Nuc-Wipes can help you maintain the safety of your working environment. Nuclean is National Diagnosticsâ&#x20AC;&#x2122; concentrated, effective, and economical solution for removing radioactivity from laboratory glassware, equipment and surfaces. Nuc-Wipes are unique pads for environmental wipe tests. Unlike filter paper, Nuc-Wipes dissolve in scintillation fluid, ensuring accurate, reproducible results. See pages 142-143 for more information.

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Product Name

Cat. No.

Size

Nuclean [pg142] NC-200 1 quart 1 gallon (1-3) 1 gallon (4+) Nuc-Wipes [pg143] NW-300 1 box of 100 (1-3) 1 box of 100 (4+)

Liquid Scintillation Products - Traditional Scintillation Cocktails for Vial Counting

Liquiscint

l l

Economical Reliable

Liquiscint is an economical, ready-to-use liquid scintillation solution designed for aqueous and organic samples. Liquiscint is intended as a general replacement for â&#x20AC;&#x153;homemadeâ&#x20AC;? toluene-Triton X-100 LSC solutions. For no more than it would cost to prepare your own solutions, Liquiscint offers the advantages of convenience and safety, as well as stringent quality control testing to ensure consistency from batch to batch.

APP L IC ATIO N S Excellent for: Low volume aqueous samples. Useful for: Samples on filter paper, polar solvents, low salt solutions, sucrose gradients, urine screening.

Storage: Liquiscint is best stored tightly capped in a cool dry area. It is stable for five years under normal conditions. Product Name

Counting discrete samples....................................................... p.156

Betafluor

Size

l l l l

Excellent for: Organic solvents, oxidation counting, carbon dioxide, radon. Counting discrete samples....................................................... p.156

For Non-Aqueous Samples High Counting Efficiency Ready-To-Use Economical

Betafluor is a pre-mixed scintillation solution designed to count virtually all nonaqueous and organic samples with greatly improved efficiencies. Betafluor contains all necessary primary and secondary fluors and solvents. It is precisely quality controlled to assure lot-to-lot consistency and reproducibility of results. Simply combine the nonaqueous sample with 10-15 ml Betafluor, shake well and count.

Typical Efficiencies for 10ml Betafluor and 10 ml Conventional Toluene-Based Scintillator

LSC

Betafluor has lower backgrounds and improved quench resistance over toluene-based scintillators, and tritium efficiencies over 55% are obtainable. Betafluor is formulated with our exclusive high flash point, low evaporation rate LSC solvent which offers higher counting efficiencies as well as reduced fire hazard and exposure to aromatic solvents.

Traditional Cocktails

APPL IC A TION S

Storage: Betafluor is best stored tightly capped in a cool dry area.

60 % Efficiency

Cat. No.

Liquiscint LS-121 4 liter (1-3) 4 liter (4 +) 20 liter drum

Product Name 40

Cat. No.

Size

Betafluor LS-151 4 liter (1-3) 4 liter (4 +)

Betafluor Toluene Scintillator

0.25

0.50

0.75

1.0

External Standard Ratio

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135

Liquid Scintillation Products - Biodegradable Scintillation Cocktails for Flow Counting

B io d e g r a d a b l e C o ck tails fo r HP LC F l o w D et ection National Diagnostics produces a complete line of ready-to-use solutions specifically designed for on-line use in HPLC flow detectors. These solutions can also be used for discrete counting of individual samples in liquid scintillation vials. All National Diagnostics HPLC scintillation cocktails are low viscosity, monophasic and non-gelling, thus guaranteeing pumpable, homogeneous samples.

Ecoscint Flow

l l l l l

APPLIC A TION S Excellent for: High sample capacity, high efficiency flow counting with minimal waste generation. Flow liquid scintillation............................................................. p.158

Nonhazardous and Biodegradable Low Viscosity Non-Gelling Ultra-High Sample Hold (1:1 Cocktail to Sample) Low Toxicity High Counting Efficiency

Formulated for multipurpose flow applications, Ecoscint Flow represents the utilization of National Diagnosticsâ&#x20AC;&#x2122; new advances in scintillation fluid chemistry to create a low viscosity cocktail. Ecoscint Flow accepts a wide range of HPLC gradients at a 1:1 ratio, providing high counting efficiency (25% 3H efficiency at 1:1 cocktail/sample). Even difficult samples such as 0.1N NaOH mix rapidly to yield a clear, nonviscous emulsion. Sample Hold (Volume of Sample per 10ml Ecoscint Flow)

LSC LSC

FlowCocktails Cocktails LSC

Hydrochloric Acid 0.1 N Sodium Hydroxide 0.1 N Sodium Chloride 0.5 M Sodium Chloride 1 M Sucrose 40% Ammonium Acetate 15 mM Ammonium Acetate 0.25 M Tris/HCl 0.05 M pH = 7 Tris/HCl 0.5 M pH = 7 Washing Buffer 1X PBS Ammonium Sulfate 0.1 M Acetonitrile: 1% HOAc 75:25% Acetonitrile: 1% HOAc 50:50% Acetonitrile: 1% HOAc 25:75% Acetonitrile: 1% HOAc 0:100% Acetonitrile: 0.1% TFA 75:25% Acetonitrile: 0.1% TFA 50:50% Acetonitrile: 0.1% TFA 25:75% Acetonitrile: 0.1% TFA 0:100% Acetonitrile: 1X PBS 75:25% Acetonitrile: 1X PBS 50:50% Acetonitrile: 1X PBS 25:75% Acetonitrile: 1X PBS 0:100% Methylene Chloride Acetonitrile Ammonium Formate 1 M

Product Name

6 mL 8 mL 10 mL 5 mL 150 ÂľL 10 mL 10 mL >10 mL 10 mL 10 mL 10 mL 7 mL 5 mL 5 mL 10 mL 7.5 mL 5.5 mL 5.5 mL > 10 mL 2.5 - 3 mL 2.5 - 3 mL 3.5 mL 8 mL All proportions All proportions 4 mL

Cat. No.

Size

Ecoscint Flow LS-288 4 liter (1-3) 4 liter (4+) 20 liter

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Liquid Scintillation Products - Biodegradable Scintillation Cocktails for Flow Counting

Monoflow 5 TM

APP L IC ATIO N S Excellent for: High sample capacity, high efficiency flow counting with minimal waste generation. HPLC gradients.

l l

Non-Hazardous and Biodegradable Moderate Sample Hold (3:1 Cocktail to Sample)

Monoflow 5 is a biodegradable liquid scintillator of low toxicity for HPLC effluents counted in flow detectors at ratios of up to 3:1 scintillator to sample. Monoflow 5 is nonhazardous and can be disposed of as normal liquid waste.

Flow liquid scintillation............................................................. p.158

Typical Efficiency (Tritium) of Monoflow 5 with Various Samples Lipids/ CH3CN

Monoflow 5 (3:1)*

CH3CN:H2O 50:50

CH3OH:H2O 50:50

2M Ammonium Formate

----

*(Ratio of Monoflow product : sample)

Product Name

Cat. No.

Size

Monoflow 5 LS-285 4 liter (1-3) 4 liter (4+) 20 Liter

Uniscint BD TM

Excellent for: High salt solutions, urine, ammonium phosphate or CsCl2 gradients, acidic samples, flow counting. Useful for: High volume aqueous samples, biological samples, plasma, RIA supernatants, glass fiber filters, sucrose gradients. Counting discrete samples....................................................... p.156 Flow scintillation counting........................................................ p.158

LSC LSC

APPL IC A TION S

Uniscint BD is a biodegradable scintillation solution especially formulated to accommodate high salt and buffer samples while still delivering efficiency. Uniscint BD has a low viscosity and is non-gelling. It is suited for use in on-line HPLC flow detectors as well as in traditional vial counting.

Flow LSC Cocktails Cocktails

Non-Hazardous and Biodegradable For HPLC Flow Detection of High Salt Samples Moderate Sample Hold (3:1 Cocktail to Sample)

Uniscint BD accommodates all concentrations of ammonium phosphate up to 2M, at a ratio of 3:1 scintillator to sample, making it the ideal choice for counting ammonium phosphate gradients from 0-2M. Other samples such as 2M ammonium formate and water are also accommodated at a 3:1 ratio. Product Name

Cat. No.

Size

Uniscint BD LS-276 4 liter (1-3) 4 liter (4+) 20 Liter

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137

Liquid Scintillation Products - Traditional Scintillation Cocktails for Flow Counting

Traditional Cocktails for HPLC Flow Detection Efficiencies (3H) of Non-Biodegradable Monoflow Solutions Lipids/ CH3CN

CH3OH:H2O 50:50

2M Ammonium Formate

Monoflow 1

----

Monoflow 2 (3:1)*

----

Monoflow 3 (2:1)*

----

Monoflow 4 (3:1)*

Monoflow 1

CH3CN:H2O 50:50

(Ratio of Monoflow product : sample)

l l

For Flow Detection of Non-Aqueous Samples High Counting Efficiency

Monoflow 1 is for use with flow detectors for organic soluble samples, such as lipids and steroids.

Monoflow 2 TM

l l

Versatile Scintillation Solution for Flow Detectors High Counting Efficiency and High Sample Hold

Monoflow 2 is a high efficiency scintillation solution for use with flow detectors. Monoflow 2 can accommodate aqueous salt or buffer solutions, or polar samples up to ratios of 3:1 scintillator to sample.

Monoflow 3 TM

High Sample Hold for HPLC Flow Detection Counting

Monoflow 4 TM

LSC

Flow Cocktails

Monoflow 3 is a scintillation solution for use with flow detectors and is specifically designed for highly aqueous samples up to ratios of 2:1 scintillator to sample. l

For Flow Detection of High Salt Samples

Monoflow 4 is a liquid scintillation solution for HPLC effluents containing high salt concentrations. Monoflow 4 is especially suited to accommodate ammonium phosphate gradients from 0-2M, at a flow ratio of 3:1 scintillator to sample. Monoflow 4 may also be used for discrete counting of highly polar samples in liquid scintillation vials. Product Name

Cat. No.

Size

Monoflow 1 LS-281 4 liter (1-3) 4 liter (4+) 20 Liter Monoflow 2 LS-282 4 liter (1-3) 4 liter (4+) 20 Liter Monoflow 3 LS-283 4 liter (1-3) 4 liter (4+) 20 Liter Monoflow 4 LS-284 4 liter (1-3) 4 liter (4+) 20 Liter

138

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Liquid Scintillation Products - Sample Oxidation

Sample Oxidation Solutions Accurate quantitation of 14CO2 released by respiration or sample combustion depends upon the efficiency of capture and scintillation. National Diagnostics supplies Carbamate and cocktail combinations suitable for all such applications. National Diagnosticsâ&#x20AC;&#x2122; reagents ensure compatibility between Carbamate and cocktail, which is critical to obtaining accurate and reproducible results.

Oxosol C14 TM

Complete Oxidizer Solution

Oxosol C14 is a complete scintillator designed to absorb and count 14CO2 produced by sample combustion. Product Name

Cat. No.

Size

Oxosol C LS-211 1 gallon (1-3) 1 gallon (4 +) 14

APP L IC ATIO N Excellent for: Carbon dioxide capture and counting. Gaseous samples. Counting 14CO2........................................................................ p.158

Carbamate-1

CO2 Absorber

Product Name

Cat. No.

Size

Oxosol 306 TM

LSC

Carbamate-1 LS-241 450 ml (1-3) 450 ml (4+)

Sample Oxidation

Carbamate-1 is a high-capacity CO2 absorber intended to be used in conjunction with Oxosol 306. One (1) ml absorbs 5.8 mMoles CO2 at saturation.

Oxidizer Solution

Oxosol 306 is a complete scintillation counting solution specifically formulated to count CO2 samples trapped in Carbamate. Product Name

Cat. No.

Size

Oxosol 306 LS-231 1 Liter 4 Liter

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139

Liquid Scintillation Products - Tissue/Gel/Filter Solubilization

Ti s s u e / Gel / F i l t er Solubilization National Diagnostics has extensive experience assisting researchers in the solubilization of tissues, gels, and filters in the preparation of samples for scintillation counting. National Diagnostics is the only company offering a biodegradable solubilizer/cocktail combination.

Biosol Bioscint TM

l TM

APPLIC A TION S Excellent for: Tissue solubilization, tissue homogenates, biological samples, blood, brain, polyacrylamide gels. Counting Tissue Samples......................................................... p.157 Samples in Polyacrylamide Gels............................................ p.158

Biodegradable Tissue Solubilizer/Scintillator Combination

When used together, Biosol and Bioscint form a nonhazardous, biodegradable tissue solubilizer and scintillation solution. The solubilizer, Biosol, is designed to be used with a variety of samples, including tissue, filters and polyacrylamide gels. Bioscint is our neutralizing scintillation solution. Used in combination with Biosol, Bioscint eliminates chemiluminescence and renders the solubilizer nonhazardous. One (1) part Biosol must be mixed with ten (10) parts Bioscint to be disposed of as nonhazardous waste under EPA 40 CFR Subpart C. Neither Biosol nor Bioscint can be disposed of separately as nonhazardous waste. Biosol is compatible with all aqueous scintillation solutions. However, only Bioscint will render the solution nonhazardous for disposal. Storage: Biosol is corrosive, and is best stored tightly capped in a cool dry area. Product Name Biosol

Cat. No.

Size

LS-310 400 ml

LSC

Solubilization

Bioscint LS-309 4 liter (1-3) 4 liter (4+)

Solusol

APPLIC A TION S Excellent for: Tissue solubilization, tissue homogenates, blood, brain, polyacrylamide gels, biological samples. Counting Tissue Samples......................................................... p.157 Samples in Polyacrylamide Gels............................................ p.158

Tissue and Gel Solubilizer

Solusol is a superior solubilizing agent which may be used with a wide variety of samples. Solusol accepts large aqueous samples and accommodates most animal and plant tissues and tissue homogenates with minimal quenching and high efficiency. Solusol has also been efficiently used to recover labeled samples from polyacrylamide gels. Simply add sample to a glass scintillation vial with a nonmetallic cap (pre-wet sample if tissue is dehydrated). Add Solusol to sample vial, allow to digest and then count. An aromatic-based scintillation solution is recommended for counting. National Diagnosticsâ&#x20AC;&#x2122; Soluscint XR (Order No. LS-314) is ideally suited for this purpose. Storage: Solusol is best stored in the freezer, tightly capped under nitrogen. Product Name

Cat. No.

Size

Solusol LS-311 450 ml (1-3) 450 ml (4 +)

140

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Liquid Scintillation Products - Tissue/Gel/Filter Solubilization

Soluscint-XR

l l

APP L IC ATIO N S Excellent for: Tissue solubilization, tissue homogenates, biological samples, blood, brain, polyacrylamide gels.

Companion Cocktail for Solusol Ideal for High Salt and Alkaline Samples

Soluscint XR is National Diagnosticsâ&#x20AC;&#x2122; complete ready-to-use scintillation cocktail specifically designed to count tissue homogenized by National Diagnosticsâ&#x20AC;&#x2122; Solusol. Additionally, Soluscint XR is also ideal as a general purpose cocktail for counting high salt and highly alkaline aqueous samples. Chemiluminescence is reduced, and counting efficiency of highly quenched samples is considerably improved. Soluscint XR is capable of incorporating an aqueous sample at 25% sample hold. Soluscint XR Sample Holds with High Salt Samples

Counting Tissue Samples......................................................... p.157 Samples in Polyacrylamide Gels............................................ p.158

Hold Max/10ml

1M Tris/HCl

1.6ml

1M NaCl

1.5ml

1M Ammonium Acetate

4ml*

2M Ammonium Acetate

8ml

1M NaOH

3ml*

*In these cases the final emulsion forms a clear gel which is perfectly acceptable for counting.

Product Name

Cat. No.

Size

Soluscint-XR LS-314 4 liter (1-3) 4 liter (4 +)

F iltron-X

Dissolves Membrane Filters TM

Excellent for: Samples on cellulose ester (cellulose acetate or cellulose nitrate) membranes. Samples on Cellulose-Ester Filters.......................................... p.157 Path of Emitted Beta Rays

Product Name

Cat. No.

LSC

APP L IC ATIO N S

Solubilization

Filtron-X is a ready-to-use liquid scintillation solution which completely dissolves Millipore -type filters (cellulose acetate, cellulose nitrate and mixed ester), solubilizing a wide variety of samples contained on the filter. Filtron-X contains all the necessary phosphors and solvents to yield counting efficiencies unattainable by any other means. The use of Filtron-X assures full 4 pi geometry counting of samples. Improvement in counting efficiency is most notable when tritium is used as the label since absorption of the emitted beta ray by the filter is completely avoided. Filtron-X will solubilize the sample assuring homogeneous counting. For a completely dry filter it may be necessary to moisten the filter with one or two drops of distilled water before placing in Filtron-X. Simply add filter to 10ml of Filtron-X, allow to stand at room temperature for 15 minutes. Shake well to assure complete dissolution of filter and sample and then count. Size

Filtron-X LS-201 4 liter (1-3) 4 liter (4 +)

Note attenuation of beta rays by filter

Emissions from Labeled Compound on Intact Filter

Emissions from Labeled Compound with Filter Dissolved

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Liquid Scintillation Products - Radiation Safety

Rad ia t io n S a f et y When working with radioactivity, nothing is more important than safety. Local, national and international regulations all require that where radioactivity is being handled, surfaces must be monitored on a frequent and regular basis. National Diagnostics has introduced a range of products to help researchers with safety protocols including a convenient and cost effective wipe test kit.

N uclean

l l l l l

APPLIC A TION S Excellent for: Removing contamination from surfaces after positive wipe tests. General laboratory cleaning. Radiation safety........................................................................ p.159

Safe and Effective Radioactive Decontamination Superior Cleaner Biodegradable pH Neutral Will Not Damage Metal Instruments

Nuclean is a concentrated, economical and highly efficient solution for safe and fast removal of radioactivity from laboratory glassware, equipment and laboratory surfaces. It is also a superior general laboratory cleaner and degreaser. In normal use, Nuclean is diluted 1:50 with water, and the glassware allowed to soak overnight and rinsed clean with distilled water. Faster decontamination is effected by increasing the concentration to 1:20 and elevating the temperature. Agitation will greatly accelerate the process. For surface decontamination, such as lab benches and tops, Nuclean should be used undiluted.

LSC

Radiation Safety

Nuclean is biodegradable and mild to the skin when diluted 1:50. Nuclean is not only more effective than chromic acid but is safer to use as well. Quart containers are supplied with a spray-head. Product Name

Cat. No.

Size

Nuclean NC-200 1 quart 1 gallon (1-3) 1 gallon (4+)

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Liquid Scintillation Products - Radiation Safety

Nuc-Wip e s

l l l l

APP L IC ATIO N S Excellent for: Routine wipe testing.

Completely Dissolving Wipes for Environmental Tests Greater Counting Efficiency Increased Reliability Safe and Easy to Use

Nuc-Wipes are dissolvable pads which assure superior environmental wipe tests. Nuc-Wipes are completely soluble in any scintillation solution, and because Nuc-Wipes dissolve completely, no emitted beta ray can be hindered or absorbed by an undissolved portion of the wipe. Nuc-Wipes allow full 4 pi counting efficiencies, thereby eliminating the possibility of lost counts due to absorption of beta rays by the filter itself.

Radiation safety........................................................................ p.159

Using intact filters for environmental wipe tests can give inaccurate, erratic results. Beta rays originating from particles on intact filters are attenuated and absorbed by the filter. Furthermore, depending on the relative affinity of the material for the solution, as material leaves the filter for the solution, counts change over time.

Conventional Wipes

Nuc-Wipes

Cat. No.

Size

LSC

Product Name

Radiation Safety

Nuc-Wipes eliminate the dependence of results on the direction of the filter paper and time. Because NucWipes dissolve in scintillation fluid, there is no intact filter to absorb or attenuate beta emissions. 4 pi counting efficiency is achieved, giving reproducible results.

Nucwipes [box of 100 wipes] NW-300 1 box of 100 (1-3) 1 box of 100 (4 +)

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Liquid Scintillation Products - Scintillation Vials

A c c e s s o ri es f o r S cin tillation Coun ting S c in tillatio n Vi a l s

l l l l

APPLIC A TION S Counting discrete samples...................................................... p. 156

Precision Molded Highest Quality HDPE Leak-Tight Seal Economical

National Diagnostics’ scintillation vials are made of high density polyethylene (HDPE), providing low permeability to the organic solvents typically used in liquid scintillation counting. These scintillation vials are manufactured as a one-piece molding with no seams, thus eliminating cracked vials, pinholes and leakage. National Diagnostics’ vials also provide excellent UV light transmission for higher efficiency. A tough, resilient sealing ring is precision-molded into the cap. This offers a secure seal and prevents leakage in general applications. National Diagnostics’ scintillation vials are designed to accommodate all conventional scintillation solutions. Additionally, using National Diagnostics’ EcoscintTM family of biodegradable, low-toxicity, nonhazardous liquid scintillation solutions improves the safety of liquid scintillation procedures.

LSC

Scintillation Vials

National Diagnostics offers three types of scintillation vials: 20ml standard vials with screw caps and 6ml or 8ml minivials with push-on/twist-off caps. The minivials with push-on / twist-off caps offer a novel closure system. The push-on/twist-off cap is a safer, more time-efficient alternative to the normal screw-on or plug cap. The push-on/twist-off cap pushes on for fast closure. This is especially helpful when processing many vials at one time. The cap then twists off as a normal screw cap for safer reopening. This eliminates the splashing caused by the removal of plug caps and avoids loss of radioactivity and/or personal contamination. The push-on/twist-off cap also eliminates the possibility of the cap popping off due to pressure buildup in the vial. The vial can be opened and closed many times both safely and easily. Product Name

Cat. No.

Size

20ml Scintillation Vials SVC-20 1-10 cases with Screw Caps (1000/case) 11+ cases

144

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8ml Scintillation Vials SVC-08 with Push-on/Twist-off Caps (2000/case)

1-10 cases 11+ cases

6ml Scintillation Vials SVC-06 with Push-on/Twist-off Caps (1000/case)

1-10 cases 11+ cases

Liquid Scintillation Products - Liquid Handling

National Diagnostics Bottle-Top Dispenser

• Spring-lock cursor design ensures fine adjustment for exact and reproducible dispensing • Easily removable PTFE piston for cleaning and smooth action • Borosilicate glass barrel protected with a transparent polypropylene sleeve • Easy to adjust calibration mechanism • Anti-drip tap

Compatible with all National Diagnostics’ scintillation cocktails, the National Diagnostics Bottle-Top Dispenser has an accuracy of delivery with 0.3% on maximum delivery and a precision better than 0.1% CV. The National Diagnotics Bottle-Top Dispenser is fitted with an anti-drip safety valve, a feature which guards against drips when the Bottle-Top Dispenser is not in use. A fine adjustment mechanism allows for even greater reproducibility for repeat dispensing. The right-angled delivery spout ensures accurate dispensing into narrow scintillation vials. Unlike other bottle-top dispensers, the National Diagnostics Bottle-Top Dispenser can be disassembled from the pedestal for thorough cleaning. Cat. No.

Size

Bottle-Top Dispenser

LS-900

1 Unit

Product Name

Cat. No.

Size

Extendable Delivery Jet

LS-904

1 Unit

USA: 1-800-526-3867 EUROPE: 441 482 646022

LSC

The National Diagnostics Extendable Delivery Jet gives versatility to your dispensing, facilitating high-throughput scintillation counting. The delivery jet stylus slots into the right-angled spout of the Bottle-top Dispenser for “hands-off” use when desired.

Liquid Handling

Extendable Delivery Jet

Product Name

145

Liquid Scintillation Products - Autoradiographic Enhancement

Autoradiography Image Enhancement Detection of low levels of radioactivity on TLC plates, polyacrylamide gels and histological slices can all be enhanced or accelerated by the use of National Diagnosticsâ&#x20AC;&#x2122; Autofluor. Autofluor is the first water soluble fluorographic enhancing reagent, providing the best efficiency of detection available, in a safer and easier to use solution.

Autofluor

APP L IC ATIO N Excellent for: Fluorographic detection of radiolabeled samples on polyacrylamide gels or TLC plates. Counting Samples on TLC Plates............................................ p. 157 Samples of Polyacrylamide Gels........................................... p. 158 Autoradiography...................................................................... p. 83

High Resolution Autoradiographic Image Intensifier l Rapid Enhancement of Low Energy Beta-Emitters Such as 3H, 14C, and 35S l For Polyacrylamide Gels, Paper Chromatography, and TLC Plates l Water-Based, Odorless, Contains No DMSO

The Autofluor procedure is the shortest and easiest procedure yet developed for enhancement and visualization of beta-emitters. In an independent test1 comparing eight different fluorographic methods for the detection of 35 S-labeled proteins in polyacrylamide gels, Autofluor was the most effective. With Autofluor, the dpm/mm2 required to half-saturate the x-ray film was 1/8 that required by autoradiography alone. Storage: Autofluor has a shelf life of at least one year stored at room temperature, out of direct sunlight. Keep from freezing. At temperatures less than 20OC precipitation of water soluble phosphors may occur. Warming to approximately 30OC will redissolve the phosphors.

LSC

Autoradiography

Autofluor represents the first water soluble scintillation phosphor to be developed and applied directly for use as an autoradiographic image intensifier. Autofluor rapidly penetrates acrylamide gel systems and maximizes energy transfer from labeled compound to phosphor. Autofluor contains no dimethylsulfoxide or acidic aromatic solvents. Therefore, the hazards of use related to these materials are eliminated. The band distortion that is associated with using nonaqueous enhancers is also eliminated.

PPO-DMSO

Autofluor

The left gel (7%, 1mm) in the illustration was dehydrated in DMSO (dimethylsulfoxide) for one hour, then impregnated in PPO-DMSO for one hour, precipitated and dried. The right gel was impregnated with Autofluor for one hour and dried. Both gels were exposed for 24 hours at -76Â°C on Kodak XR-5 X-OMat film. The single tritiated band contains 5000 dpm. Note the higher degree of resolution and band discrimination with Autofluor vs PPO-DMSO. Product Name

Cat. No.

Size

Autofluor LS-315 1 Liter (1-3) 1 Liter (4 +) Perng (1988), Analytical Biochemistry, 173, 387-392

146

USA: 1-800-526-3867 EUROPE: 441 482 646022

Liquid Scintillation Products - Scintillator Reagents

Primary Scintillators Butyl PBD

PPO - ULTRA PURE (2,5-diphenyloxazole)

Absolute fluorescence emission in cyclohexane (max): 357nm o

Assay by GLC.........................................99%

Melting Point.................................... 71-73 C

Purity by TLC.................................... One spot

Absolute Absorption...........................327nm

PPO SFC-10 25 g 100 g 500 g

Absolute fluorescence emission in cyclohexane (max): 363nm Assay by GLC........................................99% Purity by TLC.................................... One spot

Melting Point................................137-139 C Absolute Absorption.......................... 304nm

Butyl PBD SFC-20 25 g 100 g 500 g

Naphthalene - ULTRA PURE

p-Terphenyl

Absolute fluorescence emission in cyclohexane (max): 322nm Assay by GLC.........................................99% Purity by TLC................................... One Spot

ULTRA PURE (2,[4-biphenylyl]-5-[4-tert-butylphenyl]-1,3,4-oxadiazole)

Melting Point................................... 80-81°C Absolute Absorption...........................276nm

Naphthalene (scintillation grade) SFC-40 100 g 1 kg

ULTRA PURE

Absolute fluorescence emission in cyclohexane (max): 340nm Assay by GLC.........................................99% Purity by TLC................................... One Spot

Melting Point.................................212-213 C Absolute Absorption...........................276nm

p-Terphenyl SFC-50 25 g 100 g

Secondary Scintillators Bis-MSB

ULTRA PURE (1,4-bis[5-phenyloxazol-2-yl]benzene)

ULTRA PURE (1,4-bis[2-methylstyryl]benzene)

Spectroanalysis assay............................99% Purity by TLC................................... One Spot

Melting Point...............................244-246 C Absolute Absorption.......................... 358nm

BBQ

Melting Point................................ 179-181 C Absolute Absorption...........................346nm

TPB

ULTRA PURE (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one)

ULTRA PURE (1,1,4,4-tetraphenyl-1,3-butadiene)

Absolute fluorescence emission in cyclohexane (max): 477nm o

Melting Point...............................202-204 C Absolute Absorption.......................... 382nm

BBQ SFC-13 1 g 5 g

Spectroanalysis assay............................99% Purity by TLC................................... One Spot

Bis-MSB SFC-90 25 g 100 g

POPOP SFC-60 25 g 100 g

Spectroanalysis assay..……………………99% Purity by TLC................................... One Spot

Absolute fluorescence emission in cyclohexane (max): 420nm

LSC

Absolute fluorescence emission in cyclohexane (max): 410nm

Powder Scintillators

POPOP

Absolute fluorescence emission in cyclohexane (max): 455nm Spectroanalysis assay............................99% Purity by TLC................................... One Spot TPB

Melting Point...............................207-209 C Absolute Absorption...........................346nm

SFC-15

5 g

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147

LSC Concepts - Fundamentals of Liquid Scintillation Counting

Fundamentals of Liquid Scintillation Counting

1.1 RADIOACTIVE EMISSIONS

Types of Radioactive Emission / Characteristics of Useful Isotopes / Use of Isotopes in Research

1.2 MEASUREMENT OF RADIATION AND ISOTOPE QUANTITATION

Ionization Detection / Scintillation Detection

1.3 MECHANISM OF LIQUID SCINTILLATION COUNTING

The Role of the Solvent / The Role of Phosphors (Scintillators)

1.4 LIQUID SCINTILLATION SIGNAL INTERPRETATION

Patterns of Light Emission / Pulse Analysis / Counting Efficiency / Quenching

1.5 THE COMPLETE SCINTILLATION COCKTAIL 1.6 CHEMILUMINESCENCE AND STATIC ELECTRICITY 1.7 WASTE DISPOSAL ISSUES

Liquid Scintillation Counting...Making Light of the Situation

LSC

LSC Fundamentals

The chemical nature of an element is determined by its atomic number, not atomic mass. Changing the number of uncharged neutrons within the nucleus does not change the chemical behavior of most elements in any significant way. This makes possible the existence of isotopes, which are atoms of the same element with different atomic weight. Most isotopes are stable, and do not undergo any spontaneous nuclear changes. A subset of isotopes possess too few or too many neutrons to be stable. These are radioactive. Radioactive atoms spontaneously rearrange their nuclei, emitting energy or particles in the process.

148

Radioactive isotopes of common elements are extremely useful in life science disciplines, among others, because radioactive atoms can be substituted for their nonradioactive counterparts in chemical formulations. The resulting radioactive compound is easily detectable but still chemically identical to the original material. Two detection methods predominate for assaying such incorporated radioactivity. In autoradiography, labeled material is allowed to expose a photographic emulsion. Development of the emulsion reveals the distribution of labeled material. In the second detection method, the amount of radioactivity in labeled samples is directly measured, either by a Geiger counter or by a scintillation counter. In scintillation counting, the sample is mixed with a material that will fluoresce upon interaction with a particle emitted by radioactive decay. The scintillation counter quantifies the resulting flashes of light.

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LSC Concepts - Fundamentals of Liquid Scintillation Counting

1.1 Radioactive Emissions 1.1.1 Types of Radioactive Emission Radioactive decay occurs with the emission of particles or electromagnetic radiation from an atom due to a change within its nucleus. Forms of radioactive emission include alpha particles (α), beta particles (β), and gamma rays (γ). α particles are the least energetic, most massive of these decay products. An α particle contains two protons and two neutrons, and thus comprises a stable helium nucleus. α particles only weakly penetrate whatever matter they encounter. They are unable to penetrate even 10 cm of air. β particles are high energy electrons. These are produced during the conversion of a neutron to a proton in the nucleus. β particles are emitted in concert with a neutrino (Neutrinos are almost impossible to detect). The sum of the energies of the neutrino and β particle is a constant for a given isotope, and defines the maximum energy (Emax) which can be observed for any particle emitted from that isotope. Emax is approached only for particles emitted with a low energy neutrino. In practice a distribution of energies is observed, which is characteristic for the emitting isotope. γ -rays differ from α and β emissions in that γ-rays are electromagnetic radiation, not particles. γ -rays are quite penetrating, in many cases passing through up to 5 cm of lead. Additionally, γ -rays are capable of generating secondary β emission from material they pass through. An electron in the material may absorb the energy of the γ-ray, and be promoted to an excited state which is no longer bound to its nucleus. Such an electron escapes from the atom as a free β-particle.

1.1.2 Characteristics of Useful Isotopes The list of known radioisotopes is extensive, but the number of isotopes used in research is fairly small. To be useful as a label in research, an isotope must meet a restrictive set of qualifications. First of all, it must be an element that is already a part of the experimental system. For biological research, for example, isotopes of carbon, hydrogen, oxygen, and phosphorus are widely used. Alternatively, an element which can be substituted for another in the system may be used: sulfur isotopes can be used in place of oxygen, for example.

To determine the number of radioactive atoms present within a sample at a given time, use the equations above.

N0 = t =

decay constant half-life number of radioactive atoms initial number elapsed time

Energy (MeV)

Tritium (3H)

0.019

Half Life (t1/2) 12.3 yrs

Carbon ( C)

0.156

5730 yrs

Sulfur (35S)

0.167

87.2 days

Phosphorus (32P)

1.710

14.3 days

Phosphorus (33P)

0.249

25.3 days

Iodine ( I)

0.178

59.9 days

125

Table 1.1.2a

1.1.3 The Use of Isotopes in Research For many kinds of research, the utility of radioisotopes stems from their chemical identity with their nonradioactive counterparts. This allows their incorporation into “tracers”, radiolabeled components which can be followed and detected through a series of reaction steps. Tracers are invaluable in metabolic studies, where they allow the determination of the catabolic and/or anabolic fates of nutrient compounds. Animals are fed diets containing labeled molecules, such as sugars or amino acids, and the radioactivity is followed through the system until excretion or incorporation. Another use for isotopes has been protein and DNA analysis studies, where probes which bind to specific macromolecules can be radiolabeled without interfering with their activity.

Glycine [2 - 3H] ATP [α-35S]

ATP [α-32P]

Figure 1.1.3a Examples of radiolabeled biological molecules including tritiated glycine and ATP labeled with either 35S or 32P.

1.2 Measurement of Radiation and Isotope Quantitation Most research applications of radioisotopes, at some stage, require quantitation of the isotope, which is done by measuring the intensity of radiation emitted. Common nomenclature expresses this intensity as disintegrations per minute (DPM). The SI unit for radiation, the Becquerel (Bq), corresponds to 60 DPM (one disintegration per second). The curie, an earlier and still prevalent measure, is equal to 3.7 x 1010 Bq. Truly accurate measurement of DPM would require that every emission event be detected and counted, which is not possible in most situations. Additionally, naturally occurring isotopes and cosmic radiation contribute significant “background” radiation. Corrections for efficiency and background are needed to convert CPM, the counts per minute measured, into DPM, the number of decay events which actually occurred. Techniques have been developed for applying these corrections, and a great deal of research has been carried out to improve the efficiency of counting, using various detection systems. USA: 1-800-526-3867 EUROPE: 441 482 646022

LSC

λ = t1/2 = N =

Emission

Isotope

LSC Fundamentals

Useful isotopes must also have a reasonable half-life. The half-life of an isotope is a measure of the rate at which it decays to a nonradioactive state. As each nucleus emits its radiation, it eventually reaches a stable configuration which will not emit again. Thus a given quantity of isotope will eventually yield a finite total amount of radiation. Each nucleus decays independently, so the probability of a decay event occurring at any time is equal to the probability of any one nucleus decaying multiplied by the total number of radioactive nuclei present. This number changes as decay progresses, always proportional to the number of radioactive nuclei remaining. To express a rate of radioactive decay which is independent of the amount of material involved, the time required for the decay of 50% of the starting material, the half-life, is a useful quantity. Half-lives may range from milliseconds to thousands of years, a value characteristic of the particular isotope. Assuming that a 24 hour period is required for an experiment, an isotope with a half life (t1/2) of 6 hours would undergo 24 fold reduction in emissions, or a loss of 94%. Most experimentally used isotopes have t1/2 values of 10 days or more.

Radioisotopes: Decay Products and Energies

149 941

LSC Concepts - Fundamentals of Liquid Scintillation Counting

1.2.1 Ionization Detection Alpha, beta & gamma radiation all fall into the category of ionizing radiation. Alpha & beta particles directly ionize the atoms with which they interact, adding or removing electrons. Gamma-rays cause secondary electron emissions, which then ionize other atoms. The ionized particles left in the wake of a ray or particle can be detected as increasing conductivity in an otherwise insulating gas, which is done in electroscopes, ionization chambers or proportional counting chambers. These devices measure the pulse of conductivity between two electrodes when a particle or ray ionizes the gas between them. If a sufficiently high voltage is applied between the electrodes, an amplification of the signal can be obtained, and such counters can be quite sensitive. Their utility is severely limited by the fact that for most research applications only gas phase isotopes can be detected. This greatly complicates sample preparation (requiring the combustion of 14C to 14CO2, for example) and may preclude the analysis of some compounds entirely.

Figure 1.2.1a The stylized representation of an ionization chamber above shows a beta particle colliding and ionizing a neutral particle.

LSC

LSC Fundamentals

1.2.2 Scintillation Detection Some irradiated atoms are not fully ionized by collision with emitted particles, but instead have electrons promoted to an excited state. (A sub population of ionized atoms can recombine with an ion of opposite sign and also produce an excited state.) Excited atoms can return to ground state by releasing energy, in some cases as a photon of light. Such scintillation phenomena form the basis of a set of very sensitive radiation detection systems. In solid scintillation systems, a crystal of inorganic or organic material, the scintillator, is irradiated by the sample. The light emitted in response to this irradiation is taken as a measure of the amount of radioactivity in the sample. Solid scintillation is excellent for γ radiation which is highly penetrating and can cause scintillation throughout a large crystal. An advantage of these techniques is that the same crystal is used for each sample, which enhances reproducibility. Unlike ionization counting, a gas phase sample is not required. For α or β counting, however, solid scintillation has severe limitations. The crystal must be protected from contamination by the sample, which means that the α & β particles must traverse a barrier prior to reaching the scintillator. α particles in particular are severely attenuated by even 0.05mm of aluminum or copper, and so cannot be expected to reach a scintillator crystal through even the thinnest shielding. Liquid scintillation (LSC), detailed in the next section, was evolved to

provide a usable method for counting organic isotopic compounds. These materials are most often water soluble β-emitters. LSC addresses the need for convenience, reproducibility, and high sensitivity in these assays. It also offers solutions to the problem of counting aqueous (i.e. biological) samples in a nonaqueous environment.

1.3 Mechanism of Liquid Scintillation Counting By eliminating the combustion steps needed for gas phase analysis, the introduction of liquid scintillation counting (LSC) reduced the time required to analyze radioactive samples from hours to minutes. For low energy (“soft”) β emitters, LSC offers unmatched convenience and sensitivity. LSC detects radioactivity via the same type of light emission events which are used in solid scintillation. The key difference is that in LSC the scintillation takes place in a solution of scintillator, rather than in a solid crystal. This allows close contact between the isotope atoms and the scintillator, which is not possible with solid scintillation. With LSC the short path length of soft β emissions is not an obstacle to detection. Liquid scintillation cocktails absorb the energy emitted by radioisotopes and re-emit it as flashes of light. To accomplish these two actions, absorption and re-emission, cocktails contain two basic components, the solvent and the phosphor(s). The solvent carries out the bulk of the energy absorption. Dissolved in the solvent, molecules of phosphor convert the absorbed energy into light. Many cocktails contain additional materials to extend their range of use to different sample compositions, but the solvent and the phosphor provide the scintillation of the mixture.

1.3.1 The Role of the Solvent The solvent portion of an LSC cocktail comprises from 60 - 99% of the total solution. When a radioisotope dissolved in the cocktail undergoes an emission event, it is highly probable that the particle or ray will encounter only solvent molecules before its energy is spent. For this reason, the solvent must act as an efficient collector of energy, and it must conduct that energy to the phosphor molecules instead of dissipating the energy by some other mechanism. The solvent must not quench the scintillation of the phosphor, and, finally, the solvent must dissolve the phosphor to produce a stable, countable solution. Aromatic organics have proven to be the best solvents for LSC. The prototypical LSC solvent is toluene (The solvents used in National Diagnostics scintillation fluids are safer and less toxic than toluene). The π cloud of the toluene ring (or any aromatic ring) provides a target for β-interaction, which captures the energy of the incident particle. This captured energy is generally lost through transfer to another solvent molecule, as toluene has little tendency to emit light or undergo other alternate decay modes. Thus, a β-particle passing through a toluene solution leaves in its wake a number of energized toluene molecules. The energy from these molecules passes back and forth among the solvent ring systems, allowing efficient capture by dissolved phosphors.

Toluene Figure 1.2.2a In a solid scintillator, beta and alpha particles cannot penetrate the barrier between the sample well and the NaI crystal, but gamma rays pass through easily.

Pseudocumene 150

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PXE (phenyl xylylethane)

Figure 1.3.1a Solvents employed in liquid scintillation cocktails, such as toluene, pseudocumene, or PXE, possess aromatic rings to absorb the energy of incident radiation.

LSC Concepts - Fundamentals of Liquid Scintillation Counting β particle Toluene

p-Terphenyl

Figure 1.3.1b The solvent molecules in a scintillation cocktail absorb a portion of an alpha or beta particle’s energy. The energy passes between solvent molecules until the energy reaches a phosphor, which absorbs the energy and re-emits it as light.

1.3.2 The Role of Phosphors (Scintillators) Phosphors are broadly divided into two classes: primary and secondary scintillators. Included at 0.3-1% of the solution volume, primary scintillators provide the conversion of captured energy to the emission of light. The molecules of scintillator appear to induce a dipole moment in their solvation shell, allowing direct transfer of energy between the scintillator and excited solvent molecules separated by up to 10 other solvent molecules. Primary scintillators must be capable of being excited to a light emitting state by excited solvent molecules, and they must be soluble in the solvent at a sufficient concentration to give efficient energy capture. Secondary scintillators, or wavelength shifters, were originally included in scintillation cocktails to compensate for the narrow spectral response of early photomultiplier tubes. Most primary scintillators emit light below 408nm, but the response of early photomultiplier tubes drops significantly in this range. A secondary scintillator captures the fluorescence energy of the excited primary scintillator, and re-emits it as a longer wave length signal. The process by which this energy exchange takes place is not clear. (Although the emission spectrum of the primary

Primary Scintillators Scintillator

Structure

Butyl PBD

2-[4-biphenylyl]-5-[4-tert-butylphenyl]-1,3,4-oxadiazole) Order No. SFC-20

Naphthalene

Order No. SFC-40

PPO

Emission Wavelength 363nm

322nm

357nm

2,5-diphenyloxazole Order No. SFC-10

340nm

Order No. SFC-50

Secondary Scintillators 477nm

Bis-MSB

420nm

(7H-benzimidazo[2,1-a]benz [de]isoquinoline-7-one) Order No. SFC-13

(1,4-bis[2-methylstyryl]benzene) Order No. SFC-90

POPOP

410nm

TPB

455nm

(1,4-bis[5-phenyloxazol-2-yl] benzene) Order No. SFC-60 (1,1,4,4-tetraphenyl-1,3-butadiene) Order No. SFC-15

1.4 Liquid Scintillation Signal Interpretation 1.4.1 Patterns of Light Emission A β particle, passing through a scintillation cocktail, leaves a trail of energized solvent molecules. These excited solvent molecules transfer their energy to scintillator molecules, which give off light. Each scintillator molecule gives off only one photon on activation, (and the wavelength of that photon is characteristic of the scintillator, not the β-particle), but multiple scintillators are activated by the energized molecules generated by one β-particle. The path of a β-particle in a cocktail is generally less than 0.1 cm; and the half life is correspondingly short, which means that the burst of photons from an emission event derives from a small space, and reaches the PMT with sufficient simultaneity to be read as one pulse of light. The number of photons generated is directly proportional to the path length of the β particle, which is in turn determined by its emission energy (the β particle rebounds from solvent molecule to solvent molecule, until its incident energy is exhausted). The intensity of each light pulse corresponds to the emission energy and the number of pulses per second corresponds to the number of radioactive emissions.

LSC

BBQ

It has been found that linked benzene rings, rather than larger aromatic systems, generally make superior scintillators. PPO is the most commonly used primary, and Bis-MSB the most common secondary scintillator. Napthalene is somewhat unique, in that it can serve as a low efficiency scintillator and as a solvent, in concert with other organics.

LSC Fundamentals

p-Terphenyl

scintillator and the absorption spectrum of the secondary scintillator generally overlap, the kinetics of the exchange suggest direct contact rather than an emission-absorption event.) While modern phototubes are generally capable of counting the light pulses from the primary scintillator, secondary scintillators have been found to improve efficiency in many cases and are still included in most cocktails.

Figure 1.4.1a Passing through scintillation fluid, a single beta particle gives rise to multiple, nearly simultaneous emissions of light. These photons are registered by the photomultiplier tube as one pulse of energy. The magnitude of this light pulse corresponds to the number of photons.

Table 1.3.2a USA: 1-800-526-3867 EUROPE: 441 482 646022

151 151

LSC Concepts - Fundamentals of Liquid Scintillation Counting

1.4.2 Pulse Analysis

1.4.3 Quenching

The scintillation counter classifies each pulse of photons according to the number of photons in the pulse, which corresponds to the energy of the individual β emission event. Pulses are collated into channels, and the counts per minute (CPM) in each channel is recorded. Each channel corresponds to a specific range of β energies (channels are also known as counting windows), and counts with energies above or below set limits are excluded from a particular channel. The usual practice is for three channels to be selected, which divide the energy spectrum of emissions into low, medium and high energy. The lowest channel corresponds to the energy of 3H emissions, the highest to 32P. When the counts have all been collated, the researcher knows the intensity of radiation, expressed as CPM, and its energy distribution, or spectrum. CPM is proportional to the amount of isotope in the sample, and the spectrum indicates the identity of the isotope.

Quenching is the loss of counts due to sample or cocktail characteristics and may result from a variety of components in a sample. Quenchers are customarily divided into the categories of chemical quenchers or color quenchers. Chemical quenchers absorb radioactive energy before it is converted to light. Therefore, chemical quenchers reduce the number of photons generated by each β-particle. Color quenchers absorb light in the range of the wavelength emitted by the scintillator. In this case the number of photons emitted is not changed, but the number reaching the photomultiplier tube is reduced.

Within a theoretically ideal cocktail, all of the energy from each β particle would be collected and converted into light. The spectrum of emitted β energy and the DPM values could then be taken directly from the data. The highest energy emissions would be compared with the Emax (maximum emission energies) for known radioisotopes to confirm the isotope identity. Real cocktails, however, are less than 100% efficient in energy collection and conversion, especially with lower energy β emissions. This makes data interpretation somewhat more complex.

Below this energy, β-particles do not generate enough photons to be detected

Figure 1.4.3a Strong quenching can shift the majority of pulses below the threshold of detection (marked by the dashed red line).

In both types of quenching, the energy of all light pulses is reduced, and the total CPM is reduced by the number of pulses quenched to below detectable levels. This leads to an underestimate of the total counts, and thus of the isotope present. It also leads to an apparent shift in the energy spectrum of the sample.

Figure 1.4.2a A scintillation counter collating the energy spectrum of β emissions into three channels would read the majority of 3H emissions in the low energy channel, 14C in the intermediate channel, and 32P in the high energy channel.

LSC

LSC Fundamentals

1.4.3 Counting Efficiency

152

While the effectiveness of a scintillation cocktail may be expressed a number of ways, it is most often given as the percentage of emission events that produce a detectable pulse of photons, referred to as the counting efficiency. In other words, counting efficiency is equal to CPM/DPM—the ratio of counts per minute (CPM) to disintegrations per minute (DPM) expressed as a percentage. Counting efficiency varies for different isotopes, sample compositions and scintillation counters. Poor counting efficiency can be caused by an extremely low energy to light conversion rate, (scintillation efficiency) which, even optimally, will be a small value. It has been calculated that only some 4% of the energy from a β emission event is converted to light by even the most efficient scintillation cocktails. Fortunately, this number does not vary greatly across a wide range of β-energies, which avoids an additional level of complexity in signal interpretation. However, the low efficiency in energy conversion means that low energy β particles will only generate a few photons. 3H, for example, has a maximum β energy of 0.019 MeV, which at 4% scintillation efficiency will generate about 240 photons. The average emission energy is generally 30-40% of Emax, which would give 70-100 photons in this case. Most phototubes used in scintillation detection only detect 1 in 4 photons, so the average 3H β-emission event will produce only a 20-25 photon pulse in the counter. Clearly many emissions of below average energy, or emissions which lose photons due to sample characteristics, will fall below the level of a 1 photon event and will not register as a count on the instrument. The loss of CPM due to absorption of β-energy or photons by sample components is known as quenching. Quenching can easily reduce pulses below the detection limit of the counter, thus reducing the overall counting efficiency.

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Chemical quenching by water

Color quenching by organic nitrate Detection

Figure 1.4.3b Chemical quenching and color quenching can keep energy from a radioactive event from making its way through the scintillation mechanism to the photomultiplier tube. The stylized diagram above presents chemical quenching by water and color quenching by an organic nitrate as two possible obstacles to efficient counting.

LSC Concepts - Fundamentals of Liquid Scintillation Counting

Quench Correction

Various methods are available for quench correction. The most straightforward, but most laborious, is the use of an internal standard. A known amount of radioactivity, added to an unknown sample, will increase the DPM by a predictable amount. The difference between the increase in DPM observed and that expected is due to quenching, and allows the determination of counting efficiency for that sample. The drawback to the use of internal standards is that each sample must be counted twice. It is also inconvenient to add an internal standard to many vials.

lost while the emitted particle traverses the micelle, resulting in fewer photons per particle reaching the counter. The result is an effective quench, which can be corrected by the means given in the previous section. This quenching is dependent upon the size of the micelles, which in turn depends upon the ratio of sample to cocktail. It is important to use a correction curve which accounts for this volume dependence.

Many scintillation counters offer the use of an external standard to correct for quenching. After initial counting, a strong γ emission source is placed next to the vial and the sample is counted again. The γ rays cause secondary emission of Compton electrons, which scintillate in the cocktail like β particles. The counts due to sample radioactivity are subtracted, leaving only the Compton electron counts. The theoretical energy distribution of the Compton electrons is compared with the measured energy spectrum to determine the extent of quenching. The samples must still be counted twice, but nothing need be added to the vials, and the process may be carried out automatically by the counter. The analysis of the energy spectrum is commonly done by computing a “channels ratio.” The detected counts are divided into channels based on their relative energies, and the number of high energy counts (with two channels, the “B” channel) is compared to the number of low energy counts (channel “A”). The ratio, calculated as B/A or B/B+A, will change if the sample is quenched. Quenching reduces the intensity of each light pulse, so counts will appear to be of lower energy. This will shift counts from high to low energy channels, and decrease the channels ratio. In practice, a set of quenched standards is created by adding a quenching agent to reduce the CPM of an internal standard. The channels ratio of the external standard is then determined, and a correlation is established between quench and channels ratio. The channels ratio analysis may also be applied to the sample itself to determine quenching. Again, a set of quenched standards is assembled, and a known amount of radioactivity is added to each. A curve is constructed, relating CPM/DPM to B/B+A. Once this curve has been generated, the quench of any subsequent sample can be determined from its channels ratio. This quenching factor is then used to correct CPM to DPM.

1.5 The Complete Scintillation Cocktail

Ecoscint Ultra LS-270 Ecoscint Ultra is the best scintillation cocktail on the market with both ultra-high sample hold and ultra-high efficiency. (pg. 128) Ecoscint XR LS-272 Ecoscint XR achieves ultra-high sample hold without sacrificing efficiency. 10ml of Ecoscint XR can hold up to 10ml of most common aqueous samples. (pg.129) Ecoscint A LS-273 An excellent all around scintillation fluid, Ecoscint A is readily biodegradable with low odor and low toxicity. It has exceptional sample holding capability (40% water) and high efficiency. (pg.130) Ecoscint H LS-275 Ecoscint H is National Diagnostics’ highest efficiency scintillation fluid for aqueous samples. Ecoscint H delivers up to 62% 3H counting efficiency. (pg.130) Ecoscint O LS-274 Ecoscint O is a biodegradable scintillation fluid designed to count non-aqueous (organic soluble) samples. (pg.131)

Ecoscint Flow LS-288 Ecoscint Flow accepts a wide range of HPLC gradients at a 1:1 ratio, providing high counting efficiency. Even difficult samples such as 0.1N NaOH mix rapidly to yield a clear, nonviscous emulsion. (pg.136) Ecoscint LS-271 The first biodegradable scintillation fluid introduced, the original Ecoscint is an excellent all around performer at an affordable price. Provides good counting efficiency and sample hold. (pg.131) Monoflow 5 LS-285 Economical, biodegradable flow scintillator for HPLC effluents counted in flow detectors at ratios of up to 3:1 scintillator to sample. Monoflow 5 is nonhazardous and can be disposed of as normal liquid waste. (pg. 137)

LSC

Most scintillation cocktails designed for aqueous samples contain surfactants, which emulsify the sample into the organic solvent. Toluene containing the detergent Triton-X100 is a prototypical example of an emulsion cocktail (Figure 1.5a). When water is added to a solution of Triton-X100 in toluene, the detergent molecules orient to form micelles with their hydrophobic alkane chains facing out into the solvent, and their hydrophilic polyethylene glycol chains facing in, “dissolved” in a small amount of trapped water. Various other components are added to the cocktail in small amounts, which regulate the size of the micelles to maintain overall solution clarity.

Biodegradable Scintillation Fluids

LSC Fundamentals

Living creatures contain both hydrophobic and hydrophilic compounds, any of which may be labeled during the course of a radioactive experiment. As discussed in earlier sections, the best solvents for scintillation counting are the aromatic organics, such as toluene and xylene. Hydrophobic compounds can be counted directly in such solvents, but hydrophilic materials, which include many biological samples, are completely insoluble in simple cocktails. This requires the engineering of complex cocktails, capable of bringing hydrophilic sample molecules into close proximity to organic solvents and the dissolved scintillators.

Figure 1.5a: Micellar structure in a scintillation cocktail. Hydrophilic proteins (green) and water (blue) are emulsified by Triton X-100 (black). Radioactive emissions from the labeled protein must pass through the micelle to encounter the toluene solvent (brown) before energy can be passed to the primary and secondary phosphors (red) and be re-emitted as light.

Uniscint BD LS-276 Specially formulated to accommodate high salt and buffer samples while still delivering efficiency. Accepts NH4-HPO3 gradients up to 2M in concentration. Suitable for both flow or vial counting. (pg. 132)

Since the surfactants and other additives are generally less effective at energy capture than the solvent, emulsion cocktails are less efficient than pure solvent cocktails. In addition, the partitioning of the aqueous samples into micelles means that the radioactive emissions must escape from the micelle before beginning the scintillation process. Energy is USA: 1-800-526-3867 EUROPE: 441 482 646022

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Sample Capacity

Increasing the amount of water dispersed in an emulsion cocktail will increase micellar size, decreasing the energy in any given β particle when it finally escapes the micelle and begins to generate light. At some point, the amount of water added causes a micellar inversion, in which the organic solvent is surrounded by the surfactant, while the water makes up the bulk solution. Efficiency will decrease drastically at this point. The inversion process generally does not yield a clear solution. Above the cocktail sample holding capacity, the mixture is cloudy or opaque, and photons emitted within such a solution are lost to internal reflectance. Sample holding capacity is dependant upon sample composition and upon temperature. In planning scintillation counting experiments, it is crucial to ensure that the sample volume not be too close to the capacity of the cocktail. Such samples may turn opaque with a 1-2° change in temperature, and give falsely low readings. With the use of translucent plastic scintillation vials this type of artifact can be very difficult to detect.

Sample Holding Capacities of National Diagnostics Ecoscint A Sample

Capacity (ml sample /10ml cocktail)

Water (20°C)

4.5

Water (25°C)

4.0

Water (15°C)

5.0

0.05M Tris-HCl

4.5

0.15M NaCl

4.0

10% Sucrose

3.0

8M Urea

1.0

Table 1.5a

Many scintillation counters use coincidence counting to eliminate counts due to chemiluminescence. This system uses two photomultiplier tubes, generally mounted opposite each other. Because chemiluminescence only generates one photon at a time, only one photomultiplier tube will be activated. In contrast, the burst of photons from a genuine decay event will activate both photomultiplier tubes. Coincidence counting eliminates those emission events which do not appear at both photomultiplier tubes, thus eliminating chemiluminescence counts. However, coincidence counting will also cause some low energy emission events to be missed. A further source of spurious counts is static electricity. The energy from a static electric buildup can be released as a burst of light from the cocktail. In dry environments, with plastic vials and latex gloves, high levels of static can build up, sufficient to give 104 cpm or higher from an affected sample. Static is the likely cause if counts from an individual sample vary unpredictably from one measurement to the next. Static can be minimized by wiping the vials with a wet paper towel (water dissipates the static) or by wiping with an antistatic laundry dryer sheet.

1.7 Waste Disposal Issues An aspect of LSC which must be considered in experimental design, is waste disposal. Unlike solid scintillation, LSC adds components to the sample increasing the volume of radioactive material by up to 1000 fold. The components of the LSC cocktail may represent a hazard or a disposal problem in addition to the radioactivity. For many experiments, only a small percentage of the samples counted will have significant radioactivity, so disposal of the LSC is the predominating issue. Fortunately, biodegradable LSC cocktails have been developed, such as National Diagnostics’ Ecoscint fluids and Uniscint BD, which substantially reduce the difficulty of disposing of LSC waste.

1.6 Chemiluminescence and Static Electricity

LSC

LSC Fundamentals

Another commonly encountered artifact is chemiluminescence. This is caused by any chemical reaction which generates an excited product molecule, which decays to emit light. These reactions generate only a single photon, which may be quenched, or may reach the counter to register as a low energy emission event. Such reactions can generate 105-106 cpm, skewing both total cpm data and counts ratio information. Chemiluminescence is generally diagnosed by counting the samples twice with a period of about an hour between counts. As the chemiluminescent reaction consumes its substrate, the rate of photon production decreases noticeably over an hour, and will usually decrease to zero over the course of 2-24 hours. By contrast, even a short-lived isotope like 32P will decrease its emissions by only 5% over 24 hours.

Chemiluminescence counts Scintillation counts

Figure 1.6a Spurious counts due to chemiluminescence (green) dissipate over the course of hours, while the true count stays nearly constant. With the common isotopes used in life sciences research, the rate of radioactive decay is much slower than the decay of chemiluminescent reactions.

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Accessories for Scintillation Counting Scintillation Vials 6 ml SVC-06 6 ml volume, high density polyethylene (HDPE) scintillation vials are manufactured as a onepiece molding with no seams, which prevents cracking, pinholes and leakage. These vials provide excellent UV light transmission for high counting efficiency. (pg.144)

Nuclean NC-200 Nuclean is a concentrated, economical and highly efficient solution for safe and fast removal of radioactivity from laboratory glassware, equipment and laboratory surfaces. It is also a superior general laboratory cleaner and degreaser. (pg.142)

Scintillation Vials 8 ml SVC-08 8 ml volume, high density polyethylene (HDPE) scintillation vials are manufactured as a onepiece molding with no seams, which prevents cracking, pinholes and leakage. These vials provide excellent UV light transmission for high counting efficiency. (pg. 144)

Nuc-Wipes NW-300 Nuc-Wipes are dissolvable wipe test pads, soluble in any scintillation solution. Because Nuc-Wipes dissolve completely, full 4π counting occurs, eliminating the lost counts due to absorption by the filter, enhancing counting efficiency and reproducibility. (pg.143)

Scintillation Vials 20 ml SVC-20 20ml volume, high density polyethylene (HDPE) scintillation vials are manufactured as a onepiece molding with no seams, which prevents cracking, pinholes and leakage. These vials provide excellent UV light transmission for high counting efficiency. (pg.144)

LSC Concepts - Applications of Liquid Scintillation Counting

Applications of Liquid Scintillation Counting

2.1 COUNTING DISCRETE SAMPLES

Sample Neutralization (Elimination of Chemiluminescence) / Decolorizing

2.2 SPECIAL SAMPLE PREPARATION PROTOCOLS

2.3 FLOW LIQUID SCINTILLATION 2.4 LIQUID SCINTILLATION AND RADIATION SAFETY

TLC Plates / Counting Samples on Celluloseester Filters (MilliporeTM filters) / Counting Tissue Samples / Counting 14CO2 / Samples in Polyacrylamide Gels

LSC Applications...Make the Prize Light! - William Shakespeare, The Tempest

ike solid scintillation, liquid scintillation counting was originally applied only to discrete samples, either aqueous or nonaqueous, and protocols were developed for each type. The introduction of flow counting apparatus made it possible to use LSC to monitor the effluent from a chromatography column for radioactive peaks. Again, the samples in flow LSC experiments are divided between aqueous and nonaqueous compositions.

LSC

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LSC Applications

Cocktails have been developed for discrete samples and for flow applications, answering the specific needs of each type of experiment. In addition, cocktails are available for a variety of specific applications for the counting of discrete samples with unique characteristics. Most of these cocktails are designed to simplify sample preparation in such applications as counting samples in electrophoresis gels, samples on filters, whole tissue samples or combusted materials.

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2.1 Counting Discrete Samples Liquid scintillation counting of discrete samples is conceptually straightforward. A sample is mixed with an appropriate volume of scintillation cocktail, and the mixture is placed in an LSC vial and counted. For some samples, no additional steps are required, but in many situations, samples must be processed to avoid artifacts. The most common causes of artifacts are static electricity counts, chemiluminescent counts and color quenching. Protocols for sample preparation to maximize the efficiency of counting and minimize background are given below. These protocols can be readily adapted to a variety of samples. Following this section, the preparation and counting of several unique sample types are presented, as “special applications.” The best counting efficiencies are achieved when samples uniformly disperse into the cocktail to produce a clear, colorless, pH neutral emulsion. Uniform dispersion of the sample is achieved by selecting the appropriate cocktail formulation. Organic samples present no problem. With organic samples, the highest efficiency can be achieved in cocktails which contain no emulsifiers and which are only suitable for organics. Organic samples, however, can be successfully counted in emulsifying cocktails, and often the convenience of using one cocktail for all applications outweighs any loss in efficiency. Aqueous samples present more of a challenge. The choice of cocktail will depend upon the balancing of sample holding and efficiency. It is a good idea to choose a cocktail which can hold at least 10% more sample than you intend to add, as sample capacity may be strongly affected by temperature or sample components.

LSC

LSC Applications

Biodegradable Scintillation Fluids Ecoscint Ultra LS-270 Ecoscint Ultra is the best scintillation cocktail on the market. 10ml of Ecoscint XR can hold up to 12ml of water. With extremely low background, Ecoscint Ultra delivers unparalleled counting efficiency of both small and large aqueous samples. (pg.128)

Ecoscint LS-271 The first biodegradable scintillation fluid introduced, the original Ecoscint is an excellent all around performer at an affordable price. Provides good counting efficiency and sample hold. (pg.131)

Ecoscint XR LS-272 Ecoscint XR achieves ultra-high sample hold without sacrificing efficiency. 10ml of Ecoscint XR can hold up to 10ml of most common aqueous samples, easily accepting high pH, low pH, or high salt samples. (pg.129)

Ecoscint O LS-274 Ecoscint O is a biodegradable scintillation fluid designed to count non-aqueous (organic soluble) samples. Ecoscint O delivers ultra-high efficiency and extremely low background. (pg.131)

Ecoscint A LS-273 An excellent all around scintillation fluid, Ecoscint A is readily biodegradable with high flash point, low odor and low toxicity. It has exceptional sample holding capability (40% water) and high efficiency. (pg.130)

Ecoscint H LS-275 Ecoscint H is National Diagnostics’ highest efficiency scintillation fluid for aqueous samples. Ecoscint H delivers up to 62% 3H counting efficiency. It can hold up to 10% of its own volume of aqueous sample. (pg.130)

2.1.2 Decolorizing Achieving a colorless solution of sample in cocktail is generally not problematic. Many samples are colorless, or contain so little color that dilution into the scintillation cocktail gives an essentially colorless solution. In those cases where samples are deeply colored, particularly when the sample absorbs in the region of 300-400 nm, where scintillation phosphors emit, several decolorizing protocols are available. As visible color often depends upon long conjugated polyene systems, strong oxidants are used to “bleach” the samples. Samples can be treated quite harshly prior to counting, because chemical changes to the labeled compounds will not alter the number of DPM emitted. Protocol 2.1.2a Decolorizing LSC Samples with Ultraviolet Light Ultraviolet irradiation is often effective in bleaching visibly colored samples. The optimal wavelength, intensity and time must of course be determined for each sample. In many cases, exposing samples to sunlight for 1-2 hours is sufficient. Bleaching by UV has a great advantage over other methods - nothing is added to the sample, avoiding the potential quenching or chemiluminescent effects of other bleaching agents. Protocol 2.1.2b Decolorizing LSC Samples with Hydrogen Peroxide H2O2 is a strong oxidant and a very effective bleaching agent. It is inexpensive, easy to work with, and miscible with aqueous samples. The only disadvantage to using H2O2 is that it decomposes to produce molecular oxygen, which is an effective quenching agent. Samples must be heated to drive off the O2 following H2O2 bleaching, to ensure reproducible results. 1. Mix: 0.1 - 0.3 ml of 30% H2O2 with 1ml sample 2. Incubate 1 hour at 50°C, shake occasionally. 3. Cool to room temperature, add scintillation cocktail and count. Protocol 2.1.2c Decolorizing LSC Samples with Benzoyl Peroxide Samples which are not soluble in water or tissues which have been dissolved in organic solubilizers, can be bleached with Benzoyl Peroxide. 1. Dissolve 1g Benzoyl Peroxide in 5 ml Toluene - heating to 60°C may be required. Filter solution if cloudy. (Caution: Toluene has a flash point of 7°C. Heating must be carried out in a spark-free fume hood to avoid an explosion hazard.) 2. Add 2 ml Benzoyl Peroxide/Toluene solution to 1 ml sample. 3. Incubate for 30 minutes at 50°C. 4. Cool to room temperature, add scintillation cocktail and count.

2.1.1 Sample Neutralization (Elimination of Chemiluminescence) The neutralization of strongly alkaline samples is necessary to avoid chemiluminescence (Section 1.6). Neutralization can be accomplished by the addition of acetic acid. If the sample contains a high concentration of alkali, the addition of acetic acid may increase the overall salt content beyond the capacity of the scintillation cocktail. In such situations, the sample will need to be diluted prior to the addition of cocktail. If 156

neutralization is not practical, samples may be left to stand 1-3 hours, or in some cases, overnight, before counting. This allows time for the chemiluminescent reaction to run its course and die out. If chemiluminescence is suspected, samples should be counted repeatedly at intervals of greater than 1 hour until a stable reading is obtained.

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2.2 Special Sample Preparation The preceding sections have outlined LSC procedures for samples which require only minimal preparation prior to counting. Often it is necessary to count materials which are not well suited to LSC. The problem is usually one of counting geometry, which is related to sample dispersion. Figure 2.2a (pg. 157) shows the difference between the counting geometry of a well-dispersed sample vs. one adhering to filter paper. Counts are lost to absorption because they are emitted in a direction which does not take them into the cocktail. Additionally, in a sample which rests on the bottom of the tube fully 50% of the counts may be

LSC Concepts - Applications of Liquid Scintillation Counting lost, as any downward emissions will either fail to scintillate or will generate photons with no available path to the PMT. Examples of samples which present dispersion problems are silica particles from TLC plates, precipitates collected on filters, tissue samples, and polyacrylamide gels. Protocols are provided here for these samples.

Protocol 2.2.2a Counting Samples on Cellulose-Ester Filters Do not dry the filters, because this will slow the dispersion process. If a filter has dried, dampen it with 1-2 drops of distilled water. 1. Place damp filter with sample into 10ml of National Diagnostics’ Filtron X.

2.2.1 TLC Plates

2. Allow to stand at room temperature for 15 minutes. Shake well and count.

In a typical TLC experiment, the radioactivity is detected at two points: after TLC it is analyzed by autoradiography (Electrophoresis Theory, Section 4.1.3), to locate radioactive spots. These spots are then scraped off of the plate and counted to provide quantitative information. Each of these steps can be enhanced using the following protocols. Protocol 2.2.1a Autoradiography and LSC with TLC Plates A. Autoradiography (Fluorography) After the plate has been developed, spray twice with National Diagnostics’ Autofluor and allow the plate to dry. This impregnates the plate with phosphors, which will convert the β emissions to more readily detectable photons. Autofluor enhances the speed and sensitivity of detection when the plate is placed on film. For details on autoradiography procedures, see Electrophoresis Theory, Section 4.1.3. B. Liquid scintillation counting of scraped TLC silica

Counting Samples on Cellulose Ester Filters Filtron-X LS-201 A complete scintillation fluid Filtron-X solubilizes cellulose acetate, cellulose nitrate and mixed ester filter disks assuring homogeneous counting. (pg.141)

2.2.3 Counting Tissue Samples Samples of animal or plant tissue are rarely thin or small enough to allow for full counting efficiency. Homogenization of such samples will allow them to be dispersed into a cocktail, but processing large numbers of radioactive samples by homogenization is not practical. To allow for efficient and consistent counting of tissue samples, tissue solubilizers have been developed. These products contain strong denaturants and other agents, which can dissolve tissues at moderately elevated temperatures.

Option 1: Suspend the silica powder in 10ml of a cocktail which has a gel phase with water such as National Diagnostics’ Hydrofluor. Shake well, and add 3ml H2O. Shake until gel forms, and count. Option 2: Suspend the silica in 10ml of an organic based (non-emulsion) cocktail such as National Diagnostics’ Ecoscint O. Add 0.5-1g finely divided silica thixotrophic agent to form a clear gel in the solution, keeping the TLC particles suspended. Count as usual.

Protocol 2.2.3a Counting Tissue Samples BIOSOL/BIOSCINT 1. Place up to 200 mg of tissue, or 1 ml of blood, in a glass scintillation vial. Ground or minced tissue will dissolve more rapidly. Avoid adhesion of the sample to the bottom of the vial, as this will extend the digestion time. 2. Add 1 ml of Biosol. Agitate gently (do not vortex).

Autoradiography and Scintillation Counting with TLC Plates Autofluor LS-315 National Diagnostics’ autoradiographic image intensifier, Autofluor, is a water based phosphor yielding superior results to PPO-DMSO. (pg. 146)

Hydrofluor LS-111 High performance scintillation fluid with a traditional solvent base, Hydrofluor offers a gel phase option for counting particulate samples. (pg. 133)

3. Incubate in shaking water bath at 50°C for 1-4 hours, until clear. 4. If necessary (for blood or other pigmented samples) decolorize with 0.2 ml of 30% H2O2. Cap loosely and incubate at 50°C 1 hour. 5. Cool to room temperature, add 10 ml of Bioscint and count. SOLUSOL The measurement quantities below are scaled for counting in 20ml scintillation vials. They may be scaled down for smaller vials.

Ecoscint O LS-274 Ecoscint O is a biodegradable scintillation fluid designed to count non-aqueous (organic soluble) samples. (pg. 131)

1. Place up to 200mg of tissue, or 1 ml of blood, in a glass scintillation vial. Ground or minced tissue will dissolve more rapidly. Avoid adhesion of the sample to the bottom of the vial, as this will extend the digestion time.

Figure 2.2a : The well-dispersed sample on the right achieves 4π counting geometry, while half of the counts with the sample on the right are lost due to absorption and attenuation of emissions by the filter paper.

3. Incubate at 50 C for 1-2 hours, or at room temperature for 3-5 hours, until clear. 4. Add 1ml of methanol to make the bubbles disappear. 5. If necessary (for blood or other pigmented samples) decolorize by adding 3 drops of H2O2 and leave samples at room temperature for one hour. 6. Add 15ml of Soluscint XR and leave overnight before counting.

LSC

A common radiotracer technique is to precipitate macromolecules (protein & DNA) with TCA or some other strong denaturant, collect the precipitate on a filter and count it. Often such procedures give variable results, depending upon the degree to which the sample disperses from the filter into the cocktail. A typical artifact is counts which rise over time as more material dissolves off of the filter. It is possible to avoid these artifacts by using a cocktail which dissolves the filter, reproducibly releasing all of the sample for counting.

2. Add 1ml of Solusol. Agitate gently (do not vortex).

LSC Applications

2.2.2 Counting Samples on Cellulose-Ester Filters

Products for Counting Tissue Samples Biosol LS-310 Bioscint LS-309 Together, Biosol and Bioscint form a nonhazardous, biodegradable tissue solubilizer and scintillation system. The combination eliminates chemiluminescence and renders the mixture nonhazardous. (pg.140)

Solusol LS-311 Soluscint XR LS-314 Solusol is National Diagnostics’ traditionally formulated solubilizer. Soluscint XR is the scintillant designed for use with samples solubilized by Solusol. (pp. 140-141)

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2.2.4 Counting 14CO2

4. Place gel directly onto filter paper and dry under heat (80째C) and vacuum. 5. Expose at -80째C. 24 hours is generally sufficient for 14C or 3H samples, although up to 72 hours may be required for maximum detection of 3H.

Prior to the introduction of liquid scintillation counting, a primary route of radiotracer analysis was to combust the organic material and detect the 14CO2 so generated in a gas phase proportional counter. Many protocols still call for combustion and 14CO2 counting, and many metabolic studies require quantitation of 14CO2 exhaled by tracer-fed animals. 14CO2 is assayed by trapping it in a liquid phase as a complex with a strong base (carbamate) and then counting the liquid phase.

DISSOLUTION AND COUNTING OF GEL SLICES 1. Using the autoradiography film as a template, cut out the band(s) of interest. 2. To every 100mg of gel, add 0.5ml 30% H2O2. 3. Incubate in a LSC vial at 50째C until digested (1-4 hours). 4. Heat at 37째C for one additional hour to drive off residual O2. Figure 2.2.4a: Bubbling 14CO2 through carbamate traps the gas in the liquid phase, yielding a suitable sample for liquid scintillation counting.

Protocol 2.2.4a Counting CO2 14

OXOSOL C14 This is a single solution containing both carbamate and scintillators. Gas containing 14CO2 is shaken with the cocktail or bubbled through with a sparger. It is advisable to extract the gas with a second volume of solution to ensure capture of >90% of the 14CO2 . Cap the vials and count. CARBAMATE-1 + OXOSOL 306 In this system, the carbamate is provided as a separate solution, which may enhance capture efficiencies. Shake or bubble the gas with 1ml of carbamate. Add 10ml of Oxosol 306 and count.

5. Cool, add 10ml of a scintillation cocktail capable of holding 0.6ml of aqueous material (such as Ecoscint H) and count.

2.3 Flow Liquid Scintillation Radiolabeled materials are often analyzed by chromatography. The original application of liquid scintillation counting to chromatographic techniques was to collect and count discrete fractions. This manner of counting is extremely laborious, and resolution is limited by the size of the fractions collected. Flow detectors were introduced to allow continuous LSC monitoring of column effluents. This gives extremely high resolution results that are simultaneous with the separation. Modern flow detectors also have a switchable outflow, allowing radioactive peaks in the chromatogram to be collected for further analysis, or simply to sequester radioactive from nonradioactive waste.

Scintillation fluid

from HPLC

Products for Counting 14CO2 Oxosol C14 LS-211 Oxosol C 14 is a complete scintillator designed to absorb and count 14CO2 produced by sample combustion. (pg.139) Oxosol 306 LS-231 Complete scintillation counting solution specifically formulated to count 14CO2 samples trapped in Carbamate. (pg.139)

Carbamate-1 LS-241 Carbamate-1 is a high-capacity CO2 absorber intended to be used in conjunction with Oxosol 306. One (1) ml absorbs 5.8mM CO2 at saturation. (pg.139)

Flow cell

Pump

Waste

LSC

LSC Applications

2.2.5 Samples in Polyacrylamide Gels Complex radioactive samples are often fractionated on polyacrylamide gels. Analysis of radiolabeled samples in electrophoretic gels follow the same pattern as that on TLC plates. The gel is analyzed as a whole for radioactive bands, which are then excised and counted to obtain quantitative results. Autofluor, described in Section 2.2.1 for TLC plates, is also excellent for enhancing autoradiography of PAGE Gels (Electrophoresis Theory, Section 4.1.3). Once bands are located and excised, they can be dissolved using hydrogen peroxide and then counted efficiently. Protocol 2.2.5a Counting Samples in Polyacrylamide Gels AUTOFLUOR FLUOROGRAPHY OF ELECTROPHORESIS GELS 1. Stain and fix gel as usual. 2. Rinse gel for 15 minutes in deionized water to remove fixative. 3. Immerse the gel in Autofluor. Agitate gently for 30 minutes per mm of gel thickness. Pour off the Autofluor and retain for future use. LABEL AS RADIOACTIVE MATERIAL!

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PMT 2 PMT 1

Figure 2.3a HPLC Flow Detector

c o n t i n u e d

Mixing chamber

Coincidence filter Ratemeter circuit Data System Chart Recorder

Flow detectors operate by passing a mixture of column effluent and scintillation cocktail through a transparent or translucent channel, which is monitored by a photomultiplier tube. The flow rates of effluent and cocktail are metered to provide a constant ratio, and the channel (generally a plastic tube) is coiled to cover the entire window area of the PMT, (see figure 2.3a). Cocktails for flow LSC must provide for high efficiency, high sample capacity, and low viscosity. High efficiency allows for more sensitive detection, and minimizes any decrease in signal due to sample composition or detector geometry. The sample capacity is important because flow LSC tends to generate surprising amounts of waste materials. A typical HPLC flow rate is 1ml/ min. Over an 8 hour day, continuous use of such a system will generate 1 X 60 X 8=480ml of spent solvent. If a low capacity cocktail is used (one which requires 10 volumes of cocktail per volume of sample), up to 5 liters of waste will be produced. Use of a high capacity cocktail (3:1 or 2:1) will reduce this amount by up to 70% and keep disposal costs under control. Because of the large amounts of cocktail used, switching of labeled peaks to a separate waste collection and using a biodegradable cocktail are recommended.

LSC Concepts - Applications of Liquid Scintillation Counting Applications of National Diagnostics Flow Scintillation Cocktails Cocktail

Sample Capacity (ml /10ml cocktail)

Biodegradable

Applications

Ecoscint Flow

All purpose scintillation fluid for a wide range of sample types. Ultra-high sample hold.

Monoflow 1

N/A

Organic effluents. Lipid and steroid separations.

Order # LS-288

Order # LS-281

Monoflow 2

Monoflow 3

Monoflow 4

High salt aqueous samples. Can accommodate 2M salt gradients.

Monoflow 5

Biodegradable cocktail for routine low salt aqueous samples (<200mM salt)

Uniscint BD

Order # LS-282

Order # LS-283

Order # LS-284

Order # LS-285

Order # LS-276

Routine low salt aqueous effluents (<200mM salt) Routine low salt aqueous effluents. Higher sample holding capacity than Monoflow 2

Biodegradable cocktail for high salt aqueous samples. Can accommodate up to 2M salt gradients.

exposed by the emissions which affect the worker. A correlation between film exposure and worker exposure allows the detection of dangerous levels of radiation in the lab. Radiation safety departments also use film badges to ensure that no user exceeds their long term exposure limits in a given year. Short term monitoring of exposure to high-energy emissions can be done with a Geiger counter, which detects the ionization of a gas in a sealed tube. Only those emissions which are energetic enough to penetrate the tube can be detected: 32P and the highest energy emissions from 35S. Geiger counters can be invaluable for checking gloves for contamination during the course of an experiment.

Wipe Testing

Once an experiment is finished, a comprehensive and sensitive check of all work areas is required. This is accomplished by use of “wipe tests.” A 4 cm2 piece of paper or other absorbent material is rubbed vigorously over the work area, placed in scintillation cocktail and counted. If counts above background are detected, the contaminated area is subdivided and the divisions wipe tested. Contaminated areas are cleaned and retested until no contamination can be detected. The type of filter used in wipe testing has a marked effect on the reliability of the results obtained. Often standard filter paper discs are used. Such discs generally adhere to the side or the bottom of the scintillation vial. If the vial is placed in the counter such that the filter is on the side facing the photomultiplier tube, much of the light emitted by the cocktail will be absorbed by the filter. This will give artificially low numbers of counts, measuring contaminated areas as clean. This hazard is avoided by the use of a wipe which dissolves in the scintillation cocktail such as National Diagnostics’ Nuc-Wipes.

Table 2.3a

2.4 Liquid Scintillation and Radiation Safety Working with radioactive isotopes requires diligent attention to safety measures, in order to avoid hazardous exposure(s). Because radioactivity cannot be detected without instrumentation, spills can easily be spread through and even out of the lab before they are noticed. Safety in radioisotope work requires sufficient attention to both containment and surveillance. Containment measures are designed to prevent the release of isotopes in unprotected areas. Surveillance aims to detect such accidental releases as rapidly as possible to prevent the spread of contamination.

Surveillance begins with personal dosimeters and Geiger counters. Dosimeters are most often film badges and film rings. These are worn during radioactive experiments, and the film contained within them is

Efficient and effective cleaning of spills requires some knowledge of the chemical nature of the labeled compound. Water soluble materials will come off of nonabsorbent surfaces with detergent solutions. Hydrophobic compounds require the use of higher detergent concentrations. Extremely hydrophobic compounds or absorbent surfaces may require the use of organic solvents. If an unknown sample is spilled (spent culture medium, cellular extracts, etc.), a general purpose radioactive decontamination agent should be tried, such as National Diagnostics’ Nuclean, followed by solvents if necessary.

LSC

Containment measures are taught in radiation safety courses. A brief summary is presented here, which is not intended as a substitute for such a course. The primary safeguard against radioactive spills is common sense. Radioactivity should be handled only in designated areas, which should be covered with disposable absorbent pads. Droplets of radioactive solutions will be absorbed and trapped by the pads, which are then disposed of as solid radioactive waste. Users of radioactivity must wear lab coats, gloves and eye protection. Gloves should be monitored with a Geiger counter if the isotope emissions are detectable by this means. In any case, frequent changing of gloves if contamination is detected or suspected will keep exposure to a minimum. Shielding should be used for 125I and 32P work, or for any isotope whose emissions can penetrate skin, but it is important to keep in mind that the primary long term danger from radioactive components is through ingestion or skin absorption. Eating or drinking or even chewing gum must be excluded from radioactive use areas.

Figure 2.4b Nuc-Wipes eliminate the dependence of results on the direction of the filter paper and time. Because Nuc-Wipes dissolve in scintillation fluid, there is no intact filter to absorb or attenuate beta emissions. 4π counting efficiency is achieved, giving reproducible results.

LSC Applications

Containment and Monitoring

Figure 2.4a Using intact filters for environmental wipe tests can give inaccurate, erratic results. β particles originating from particles on intact filters are attenuated and absorbed by the filter. Furthermore, depending on the relative affinity of the material for the solution, as material leaves the filter for the solution, counts change over time.

Products for Radiation Safety Nuc-Wipes NW-300 Nuc-Wipes are dissolvable wipe test pads, soluble in any scintillation solution. Because Nuc-Wipes dissolve completely, full 4π counting occurs, eliminating the lost counts due to absorption by the filter, enhancing counting efficiency and reproducibility. (pg.143)

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159 951

LSC Concepts - Troubleshooting

Sample Overload

Exceeding the sample holding capacity of a scintillation cocktail will yield unpredictable results. The mixture will become opaque, with most photons being lost to internal reflectance. Phase separation may occur, which can increase or decrease counts depending on how the sample partitions. 1) Sample composition: The salt content, pH, protein content, etc., of a sample will determine the amount of cocktail needed to provide a clear, countable emulsion. Samples above 0.5M salt, or with pH < 4 or pH > 10 will generally require more cocktail. 2) Temperature: Sample/cocktail emulsions have a range of temperature in which they remain clear and countable. This range narrows as the sample volume approaches the cocktail holding capacity. The heat of mixing associated with adding sample to the cocktail can cause sample/cocktail mixtures which are initially clear to cloud on standing, leading to a drop in counting efficiency. Addition of more cocktail will restore a clear solution. 3) Phase separation: The opaque solution generated by sample overload may separate into two phases on standing. If the sample molecules partition into the organic phase, this can increase efficiency. However, because this is a slow process, the effect is unpredictable, and manifests as erratic counting from sample to sample. 4) Standard curves: It is important to plan standard curves so that the sample capacity is not approached, to ensure that the efficiency is the same for all points.

Counting Geometry

Particulate samples, or samples bound to a solid support, will have some emission events which are absorbed by the solid before reaching the cocktail. This can reduce efficiency by as much as 50%. 1) Particulate samples: Insoluble powders must be suspended in a cocktail which forms a gel, to avoid changes in counts as the particles settle. 2) Samples on solid support: For TLC plate scrapings, see (A) particulate samples, above. Samples bound onto modified cellulose filters, use Filtron-X to dissolve the filter. For glass fiber filters, use a cocktail which will dissolve the sample components, and allow sufficient time and mixing for complete elution. Be sure filter is not in the path of the PMT.

T r ou b l e s hooti ng L i q ui d Sci n t i l l a t i on E x p e ri m e n t s

LSC

Troubleshooting

Symptom

160

Diagnosis

Counts increase with time

Sample not fully separated from solid support (B-2) or solid sample not fully dispersed/dissolved in cocktail (B-1).

Counts decrease with time

Chemiluminescence (C-1) or sample overload (A-2,3).

Erratic counting- recounting gives inconsistent results

Static electricity (C-2), sample overload (A-2,3), or solid sample settling out of cocktail (B-1).

Low efficiency- liquid samples

Color Quenching (D-1), chemical quenching (D-2), or sample overload (A-1).

Low efficiency- solid samples

Sample not fully dispersed or dissolved (B-1).

Low efficiency- sample on filter

Sample not eluted from filter (B-2), filter blocking PMT window (B-2), or quenching (D-1,2).

Standard curve not linear

Sample overload (A-4).

Chemiluminescence and Static Electricity

Light emission from cocktails can be stimulated by static electricity or chemically excited molecules. Such emissions are relatively short lived. 1) Chemiluminescence: Caused by reactions which generate chemically excited products, its lifetime is limited by the amount of substrate in the sample. Samples should be recounted repeatedly until a stable result is obtained. Alkaline samples are particularly susceptible; neutralization can minimize the problem. 2) Static electricity: Static buildup, particularly on plastic vials, can cause bursts of light emission, giving wildly erratic counts. Run water over the vial, or wipe with an antistatic dryer sheet.

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Quenching

Scintillator light emissions can be absorbed by colored sample components (color quenching). In addition, the energy from radioactive emissions can be trapped by sample components before it reaches the cocktail phosphors (chemical quenching). In both cases, total counts are reduced, and the ratio of high to low energy counts is decreased. 1) Color quenching: Samples which absorb light at 350-450nm will attenuate the light emitted from the phosphors. In general, samples which appear yellow or brown will be quenched to some extent. Quenching shifts counts from high to low energy- a shift in the ratio of high and low channels is diagnostic. Samples can be bleached with hydrogen peroxide, taking care to remove the residual oxygen, which is a chemical quencher (see (2), below). 2) Chemical Quenching: Many chemicals are able to intercept the energy from a radioactive emission event before it can be converted to light. The effect is the same as color quenching: lower counts, and proportionally more low energy counts. Water can be a chemical quencher. To ensure consistent efficiency, all samples should have the same amount of water added. Molecular Oxygen is another quenching agent, it can be removed by warming the sample to degas it. Organic compounds containing oxygen (i.e. aldehydes and alcohols), or halogens (e.g. chloroform) are generally strong chemical quenchers. Such samples should be counted in as dilute a solution as possible, to minimize the quenching effect.

LSC Concepts - Troubleshooting

Useful Information for Liquid Scintillation Counting

Radioisotopes: Decay Products and Energies Energy (MeV)

Radioisotopes: Range of Emissions Maximum Range in material (cm) Isotope

Air

12.3 yrs

Tritium (3H)

0.6

0.156

5730 yrs

Carbon (14C)

0.28

0.23

0.167

87.2 days

Sulfur (35S)

0.3

0.25

Phosphorus ( P)

1.710

14.3 days

Phosphorus (32P)

800

0.8

Phosphorus ( P)

0.249

25.3 days

Phosphorus ( P)

0.6

0.5

Iodine (125I)

0.178

59.9 days

Isotope

Emission

Tritium (3H)

0.019

Carbon ( C)

Sulfur (35S)

32 33

half life (t1/2)

Water

Plexiglass

Units of Radioactivity

Half-life (t1/2) Calculations

1 Becquerel(Bq)

= =

t1/2(half life) = the time required for 1/2 of the atoms present to undergo decay.

1 Curie(Ci)

3.7 X 1010 disintegrations per second 2.2 X 1012 disintegrations per minute

= =

1 disintegration per second (dps) 60 disintegrations per minute (dpm)

λ (decay constant) = 0.693/t1/2 N = N0e-λt where: N= number of atoms remaining at time t N0=number of atoms at start (t=0)

Suggested Reading in Liquid Scintillation Counting

Types of Radioactive Emission

Browne, E. and Firestone, R.B. (1986) Table of Radioactive Isotopes. V.S. Shirley, ed. Wiley, NY. Weast, R.C. (1988) Handbook of Chemistry and Physics 69th edition. CRC Press, Boca Raton.

Slack, L. and Way, K. (1959) Radiations from Radioactive Atoms in Frequent Use. U.S. Atomic Energy Commission Report.

LSC

Schimel, David S. (1993) Theory and Application of Tracers. Academic Press. Knoche, H.W. (1991) Radioisotopic Methods for Biological and Medical Research Oxford University Press, NY. Slater, R.J. (ed) (1990) Radioisotopes in Biology - A Practical Approach. Oxford University Press. Coleman, David C and Fry, Brian, (eds) (1991) Carbon Isotope Techniques. Academic Press. Ehmann, Wm. D. and Vance, Diane E. (1991) Radiochemistry and Nuclear Methods of Analysis. J. Wiley & Sons.

Useful Information

General Resources

Ionization Detection

Wilkinson, D.H., (1950) Ionization Chambers and Counters. Cambridge University Press, Cambridge, UK.

Liquid Scintillation Counting

Horrocks, D.L. (1974) Applications of Liquid Scintillation Counting. North Holland, Amsterdam Birks, J.B. (1964) The Theory and Practice of Scintillation Counting. Pergammon Press, Oxford, UK.

Liquid Scintillation Signal Interpretation

Verrezen, V. and Hurtgen, C., (2000) A multiple window deconvolution technique for measuring low-energy beta activity in samples contaminated with high-energy beta impurities using liquid scintillation spectrometry. Appl. Rad. and Isotopes 53 (2000) 289-296.

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161

Electro-Optical Grade Solvents

Electro-Optical Grade Solvents To reduce the environmental impact and occupational health and safety hazards associated with toxic organic clearants in the laboratory and in industry, National Diagnostics developed its original, award-winning OptiClearTM cleaning solvent. As a result of ever growing demand for safe products for cleaning optical and electronic components, National Diagnostics expanded the OptiClear product line to include an array of lower hazard solvents.

The OptiClear Solvents TM

OptiClear OptiClear OptiClear OptiClear OptiClear OptiClear R E S S2 W

OE-101 OE-102 OE-104 OE-105 OE-107 OE-106

Flash Point 120oF 142oF 45oF 147oF 177oF 199oF Non-Toxic Biodegradable Food Grade Evaporation Rate

0.4

(N-Butyl Acetate = 1)

2.0

1.6

0.04

0.0

0.06

< 5µS/cm

No Residue Water Miscible Low Odor citrus Non-Conductive

< 5µS/cm

pH (aqueous extraction) Neutral Neutral Neutral Neutral Neutral Neutral Water Content (ppm)

< 50

N.A.

< 100

< 500

Kauri-Butanol

62.7

> 200

Color APHA

< 50

Boiling Point 350oF 180oF 244-284oF 376-401oF 424-484oF 396oF Refractive Index Vapor Pressure (mm Hg)

6 @ 77 F 0.84 @ 25 C o

Viscosity (cps)

6 @ 25 C

Solids > 0.2µm Abrasives Electro-Optical Solvents

Specific Gravity

1.34 1.4041 1.4276 1.4372 1.469 o

36 @ 68 F

0.72 @ 16 C

58 @ 100 F 0.94 @ 25 C

0.5 @ 25 C

6 @ 25 C

0.1 @ 68 F

0.79 @ 16 C

1.0 @ 68 F 0.77 @ 16 C o

2.0 @ 25 C

0.5 @ 77 F

1.026 @ 25 C

3.4 @ 25 C

1.5 @ 25 C

Nil Nil Nil Nil Nil Nil None None None None None None Wax, Pitch, Flux Some Inorganic Pitch, Flux, Pitch, Flux Pitch, Flux, Flux, Oil Grease, Tar, Salts, Oil Oil, Grease Oil, Grease Oil, Grease Grease Polystyrene Grease

Dissolves

OptiClear Sample Kit I 1 Liter of Each of

162

1.47

OE-108

OE-101, OE-102, OE-105, & OE-106

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Sample Kits

Sample Kit II 1 Liter of Each of

OE-109

OE-101, OE-102, OE-104, OE-105, & OE-106

Electro-Optical Grade Solvents

OptiClear

l l l l

Non-Toxic Electro-Optical Grade Biodegradable Non-Ozone Depleting

OptiClear is National Diagnostics’ original, award winning, nontoxic, nonflammable, biodegradable solvent. OptiClear is intended for the removal of wax, pitch, flux, grease, resin and solvent soluble resists from optical and electronic components of glass, ceramic, or metal. OptiClear may be substituted directly for toluene, xylene or chlorinated hydrocarbons, such as trichloroethylene (TCE), in batch-type or ultrasonic cleaners with no alteration in protocol. OptiClear removes wax adhered to lenses, optical components, polished wafers or printed circuit boards.

APP L IC ATIO N S Dissolves Wax, pitch, grease, tar and polystyrene. Will not affect Metal and metal alloy; glass and ceramic; inorganic crystal; phenolics, epoxy, melamine, alkydes, and fiberglass. Will moderately affect Polyethylene (swells on long exposure but will not dissolve).

OptiClear has high solvency power, yet it will not affect metal or metal alloy. OptiClear also has a neutral pH, so it will not stain glass. OptiClear’s water content is less than 50 ppm. It is miscible in all proportions with acetone, ethanol, and isopropanol, any of which may be used as a post wash. Since OptiClear is 100% natural, food grade material, it is biodegradable and not categorized as a hazardous waste. METHOD 1. Use undiluted. 2. Set up an OptiClear bath at room temperature. Soak components for 15 minutes to 1 hour. For lighter cleaning applications, OptiClear can be used as a spray. 3. Remove components from bath and allow OptiClear to evaporate. 4. If excessive residue remains, rinse in OptiClear R (OE-102)

Product Name

Cat. No.

Size

Opticlear OE-101 16 oz. spray bottle 1 gallon(1-3) 1 gallon(4+) 5 gallon drum 55 gallon drum

Safety Information Note that the “nonflammable” designation of OptiClear products does not mean that the material cannot be made to burn. It means only that OptiClear products will not ignite as readily, or burn with the intensity or violence of flammable liquids such as xylene. OptiClear products are intended to dissolve paraffins and fats. Therefore, to prevent drying of the skin, avoid direct contact with the skin. Avoid contact with eyes as well as unnecessary inhalation of vapors. In case of contact with eyes, wash with large quantities of water.

Electro-Optical Solvents

With a flash point in excess of 141oF (TCC), OptiClear R, OptiClear S, and OptiClear W all exceed the requirements for classification as a nonflammable solvent by the U.S. Department of Transportation, the U.S. Environmental Protection Agency, and the National Fire Protection Association.

The information contained herein is based on the data available to us and is believed to be correct. However, National Diagnostics makes no warranty expressed or implied regarding the accuracy of this data or the results to be obtained from the use thereof. National Diagnostics assumes no responsibilities for injury from the use of the products described herein. USA: 1-800-526-3867 EUROPE: 441 482 646022

163

Electro-Optical Grade Solvents

OptiClear R TM

RINSE

OptiClear R is nonflammable, biodegradable, water-based solvent intended as an acetone replacement for cleaning optical and electronic components. OptiClear R is nonconductive, and can be used to rinse away oily film and light grease from glass, ceramic or metal without leaving any residue. OptiClear R is non-aggressive and does not attack metal, glass or plastic. Product Name

Cat. No.

Size

OptiClear R OE-102 16 oz. spray bottle 1 gallon bottle (1-3) 1 gallon bottle (4+) 5 gallon drum

OptiClear E TM

EVAPORATES FAST

APPLIC A TION S

OptiClear E is an odorless cleaning solvent for optical and electronic components. OptiClear E is our fastest evaporating OptiClear formulation, and it leaves no residue. This eliminates the need for a post wash. OptiClear E is also nonconductive. OptiClear E can be used to remove wax and pitch; however, the necessary soaking time is longer than that required for the original OptiClear. METHOD 1. Use undiluted. 2. Set up an OptiClear E bath at room temperature and soak components for 15 minutes to 1 hour. Heating is not recommended. For lighter cleaning applications, OptiClear E can be used as a spray. 3. Remove components from bath and allow OptiClear E to evaporate. 4. OptiClear E does not leave a residue, so a post-rinse is not necessary.

Dissolves Pitch, flux, oil and grease. Will not affect Metal and metal alloy; glass and ceramic; phenolics, epoxy, melamine, alkydes, and fiberglass.

OptiClear S TM

Electro-Optical Solvents

SAFE

APPLIC A TION S Dissolves Pitch, flux, oil, and grease. Will not affect Metal and metal alloy; glass and ceramic; phenolics, epoxy, melamine, alkydes, and fiberglass.

164

USA: 1-800-526-3867 EUROPE: 441 482 646022

Product Name

Cat. No.

Size

OptiClear E OE-104 16 oz. spray bottle 1 gallon bottle(1-3) 1 gallon bottle(4+) 5 gallon drum

OptiClear S is a nonflammable, odorless degreaser that removes light oil from optics and electronics components. OptiClear S is nonconductive and leaves no residue. OptiClear S can also be used to remove wax and pitch; although the necessary soaking time is longer than that required for the original OptiClear. OptiClear S has a higher flash point and evaporates more slowly than OptiClear E. METHOD 1. Use undiluted. 2. Set up an OptiClear S bath and warm to 100OF. Keep product away from sparks and flame. Soak components for 15 minutes to 1 hour. For lighter cleaning applications, OptiClear S can be used as a spray. 3. Remove components from bath and allow OptiClear S to evaporate. 4. OptiClear S does not leave a residue, so a post-rinse is not necessary. Product Name

Cat. No.

Size

OptiClear S OE-105 16 oz. spray bottle 1 gallon bottle (1-3) 1 gallon bottle (4+) 5 gallon drum 55 gallon drum

Electro-Optical Grade Solvents

OptiClear S2 TM

HIGH FLASH POINT

APP L IC ATIO N S Dissolves Pitch, flux, oil, and grease. Will not affect Metal and metal alloy; glass and ceramic; phenolics, epoxy, melamine, alkydes, and fiberglass.

OptiClear W

OptiClear S2 is a nonflammable, odorless degreaser that removes light oil from optics and electronics components. OptiClear S2 is nonconductive and leaves no residue. OptiClear S2 can also be used to remove wax and pitch; however, the necessary soaking time is longer than that required for OptiClear Original. OptiClear S2 has a higher flash point than OptiClear S but evaporates more slowly than OptiClear S. METHOD 1. Use undiluted. 2. Set up an OptiClear S2 bath and warm to 130OF. Keep product away from sparks and flame. Soak components for 15 minutes to one hour. For lighter cleaning applications, OptiClear S2 can be used as a spray. 3. Remove components from bath and allow OptiClear S2 to evaporate. 4. OptiClear S2 does not leave a residue, so a post-rinse is not necessary.

Product Name

Cat. No.

Size

Opticlear S2 OE-107 16 oz. spray bottle 1 gallon bottle (1-3) 1 gallon bottle (4+) 5 gallon drum

WATER

APP L IC ATIO N S

OptiClear W is a nonflammable, biodegradable solvent intended to remove grease and oil from optical and electronic components. OptiClear W is miscible in all proportions with water. This eliminates the need for a solvent dip, since water can be used as a post wash. METHOD 1. Use undiluted. 2. Set up an OptiClear W bath at room temperature and soak components for 15 minutes to one hour. OptiClear W can also be warmed to 140OF. For lighter cleaning applications, OptiClear W can be used as a spray. Keep product away from sparks and flame. 3. Remove components from bath and allow OptiClear W to evaporate. 4. If excessive residue remains, rinse with water.

Dissolves Flux, oil, and grease. Will not affect Metal and metal alloy; glass and ceramic; phenolics, epoxy, melamine, alkydes, and fiberglass.

Product Name

Cat. No.

Size

Opticlear W OE-106 16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon drum

Electro-Optical Solvents

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165

How to Order - USA Toll - Free Phone: 800-526-3867 Telephone: 404-699-2121 Order by Fax: 404-699-2077 Order on the Web: www.nationaldiagnostics.com E-mail: info@nationaldiagnostics.com

Index of Products

Alphabetical Product Index

166

Product Name

Cat. No.

Pack Size

Page

Product Name

Cat. No.

Pack Size

AccuGel 19:1 (30%) AccuGel 19:1 (40%) AccuGel 19:1 (40%)

EC-849 EC-850 EC-850

450 ml 1 1iter (1-3) 1 liter (4 +)

13 6 6

Ecoscint O Ecoscint O Ecoscint O

LS-274 LS-274 LS-274

4 liter (1-3) 4 liter (4+) 20 liter drum

129 184 184

AccuGel 19:1 (40%) AccuGel 19:1 (40%) AccuGel 19:1 (40%)

EC-850 EC-850 EC-850

450 ml 1 1iter (1-3) 1 liter (4 +)

13 6 6

Ecoscint Ultra Ecoscint O

LS-270 LS-274

4 liter (1-3) 4 liter (4+)

126 184

AccuGel 29:1 (30%) AccuGel 19:1 (40%) AccuGel 19:1 (40%)

EC-851 EC-850 EC-850

450 ml 1 liter (1-3) 1 liter (4 +)

8, 13 6 6

Ecoscint XR Ecoscint A

LS-272 LS-273

4 liter (1-3) 4 liter (4+)

127 182

AccuGel 29:1 (40%) AccuGel 29:1 (40%) AccuGel 29:1 (40%)

EC-852 EC-852 EC-852

450 ml 1 liter (1-3) 1 liter (4 +)

8, 13 6 6

EDTA Filtron-X

EC-610 LS-201

100 gram 500 gram

30 195

EDTA, 0.5M Solution

EC-900

1 liter

AcrylaGel AcrylaGel AcrylaGel

EC-810 EC-810 EC-810

450 ml 1 liter (1-3) 1 liter (4 +)

9, 13 7 7

Acrylamide Acrylamide Acrylamide

EC-201 EC-201 EC-201

100 gram 500 gram 1 kilogram

30 30 30

Alcian Blue

HS-504

25 gram

105

Amido Black 10B

HS-601

25 gram

21,105

Ammonium Persulfate Ammonium Persulfate

EC-504 EC-504

25 gram 100 gram

AquaPor 3:1 AquaPor ES AquaPor ES

EC-206 EC-203 EC-203

25 gram 100 gram (1-3) 100 gram (4 +)

15 9 9

AquaPor ES AquaPor ES AquaPor ES

EC-203 EC-203 EC-203

25 gram 100 gram (1-3) 100 gram (4 +)

AquaPor HR AquaPor HR AquaPor HR

EC-205 EC-205 EC-205

25 gram 100 gram (1-3) 100 gram (4 +)

AquaPor LE AquaPor LE AquaPor LE AquaPor LE

EC-202 EC-202 EC-202 EC-202

25 gram 100 gram (1-3) 100 gram (4 +) 500 gram

14 8 8 8

AquaPor LM AquaPor LM AquaPor LM

EC-204 EC-204 EC-204

25 gram 100 gram (1-3) 100 gram (4 +)

14 8 8

Autofluor Autofluor

LS-315 LS-315

1 liter (1-3) 1 liter (4 +)

28, 144 12 105

30 30

15 9 9

Basic Fuchsin

HS-518

25 gram

BBQ BBQ

SFC-13 SFC-13

1 gram 5 gram

145 201

Betafluor Betafluor

LS-151 LS-151

4 liter (1-3) 4 liter (4 +)

133 191

Biebrich Scarlet

HS-506

25 gram

105

Bioscint Bioscint

LS-309 LS-309

4 liter (1-3) 4 liter (4+)

138 194

Biosol

LS-310

400 ml

138

Bis (Methylene Bis-Acrylamide)

EC-301

25 gram

Bis-AcrylaGel Bis-AcrylaGel Bis-AcrylaGel

EC-820 EC-820 EC-820

450 ml 1 liter (1-3) 1 liter (4 +)

Bis-MSB Bis-MSB

SFC-90 SFC-90

25 gram 100 gram

145 201

9, 13 7 7

Boric Acid

EC-609

500 gram

Bottle-top Dispenser

LS-900

1 unit

143

Bromocresol Green

HS-602

5 gram

Bromophenol Blue

HS-603

10 gram

21,28,105

Butyl PBD Butyl PBD Butyl PBD

SFC-20 SFC-20 SFC-20

25 gram 100 gram 500 gram

145 200 200

Calci-Clear Calci-Clear Calci-Clear

HS-104 HS-104 HS-104

1 quart 1 gallon 5 gallon

Calci-Clear Rapid Calci-Clear Rapid Calci-Clear Rapid

HS-105 HS-105 HS-105

1 quart 1 gallon 5 gallon

100 150 150

Carbamate-1 CO2 Absorber Carbamate-1 CO2 Absorber

LS-241 LS-241

450 ml (1-3) 450 ml (4 +)

137 196

100 150 150

Coomassie Blue G-250

HS-605

10 gram

Coomassie Blue R-250

HS-604

10 gram

22 30

DATD

EC-303

25 gram

Denaturation Solution Denaturation Solution

EC-875 EC-875

1 liter (1-3) 1 liter (4 +)

19 18

Denhardt’s Solution (50X)

EC-915

50 ml

Dextran Sulfate Dextran Sulfate

EC-877 EC-877

50 gram 250 gram

18 30 31

Page

Eosin, 1% Solution

HS-402

100 ml

104

Ethanol, Reagent (Denatured) Ecoscin, REA Ecoscint A

HS-300 LS-273 LS-273

1 liter 4 liter 20 liter

30, 99

Extendable Delivery Jet

LS-904

1 unit

143

Fast Green FCF

HS-516

25 gram

105

Filtron-X Filtron-X

LS-201 LS-201

4 liter (1-3) 4 liter (4 +)

139 195

Formamide Filtron-X

EC-608 LS-201

200 ml 450 ml

30 195

GelDry Film 11 X 12 cm

EC-612

50 sheets

GelDry Film 22.5 X 22.5 cm

EC-622

50 sheets

Glass Bond

EC-620

25 ml

Glass Free

EC-621

450 ml

29 31

Glycerol

EC-606

450 ml

Glycine Glycine

EC-405 EC-405

250 gram 1 kilogram

Hematoxylin, Harris’

HS-400

1 liter

104

31 32

Histo-Clear HS-200 1liter 1 gallon (1-3) 1 gallon (4+) Histo-Clear HS-200 5 gallon Histo-Clear HS-200 55 gallon

98 148 148

Histo-Clear II Histo-Clear II Histo-Clear II

HS-202 HS-202 HS-202

1 gallon (1-3) 1 gallon (4+) 5 gallon 55 gallon

99 149 149

Histomount Histomount Histomount Histomount

HS-103 HS-103 HS-103 HS-103

100 ml (1-3) 100 ml (4+) 450 ml(1-3) 450 ml (4+)

103 153 153 153

Histosol Histosol 153 Hydrofluor Hydrofluor

HS-100 HS-100

1 gallon 5 gallon

99 149

LS-111 LS-111

4 liter (1-3) 4 liter (4 +)

131 186

Hydrogen Peroxide Assay Kit

CL-204

100 Assay Kit

121

Hydromount Hydromount

HS-106 HS-106

100 ml (1-3) 100 ml (4+)

103 153

Insite Markers

EC-897

1 Tube

Ion Exchange Resin

EC-408

100 gram

Liquiscint Liquiscint Liquiscint

LS-121 LS-121 LS-121

4 liter (1-3) 4 liter (4 +) 20 liter drum

133 190 190

Mercaptoethanol

EC-603

50 ml

MESA Buffer (10X)

EC-911

1 liter

MES-SDS Running Buffer (20X) Urea

EC-868 EC-605

450 ml 1 liter

16 33 105

Methyl Green

HS-606

10 gram

Methylene Blue

HS-525

25 gram

105

Mirsky’s Fixative Mirsky’s Fixative

HS-102 HS-102

200 ml system 2 liter system

101 151

Mirsky’s Fixative (ready-to-use) Mirsky’s Fixative (ready-to-use)

HS-101 HS-101

1 gallon 5 gallon

101 151

Monoflow 1 Monoflow 1 Monoflow 1

LS-281 LS-281 LS-281

4 liter (1-3) 4 liter (4+) 20 liter drum

138 192 192

Monoflow 2 Monoflow 2 Monoflow 2

LS-282 LS-282 LS-282

4 liter (1-3) 4 liter (4+) 20 liter drum

136 192 192

Monoflow 3 Monoflow 3 Monoflow 3

LS-283 LS-283 LS-283

4 liter (1-3) 4 liter (4+) 20 liter drum

136 192 192

Monoflow 4 Monoflow 4 Monoflow 4

LS-284 LS-284 LS-284

4 liter (1-3) 4 liter (4+) 20 liter drum

136 193 193

Monoflow 5 Monoflow 5 Monoflow 5

LS-285 LS-285 LS-285

4 liter (1-3) 4 liter (4+) 20 liter drum

135 193 193

Monofluor Monofluor

LS-191 LS-191

4 liter (1-3) 4 liter (4 +)

Diogenes

CL-202

1 kit

120

Dithiothreitol (DTT) Dithiothreitol (DTT)

EC-601 EC-601

1 gram 5 gram

Ecoscint Ecoscint Ecoscint

LS-271 LS-271 LS-271

4 liter (1-3) 4 liter (4+) 20 liter drum

129 184 184

MOPS-SDS Running Buffer (20X) Urea

EC-867 EC-605

450 ml 1 liter

16 33

Ecoscint A Ecoscint A Ecoscint A

LS-273 LS-273 LS-273

4 liter (1-3) 4 liter (4+) 20 liter drum

128 182

Naphthalene — Scintillation Grade Naphthalene — Scintillation Grade

SFC-40 SFC-40

100 gram 1 kilogram

Ecoscint Flow Ecoscint A

LS-288 LS-273

4 liter (1-3) 4 liter (4+) 20 liter drum

134

ND Protein Precipitation Kit

EC-888

1 kit

Neutralin

HS-108

5 gallon pail

102

Ecoscint GL

LS-262

4 liter

130

Neutralization Solution Neutralization Solution

EC-876 EC-876

1 liter (1-3) 1 liter (4 +)

18 18

Ecoscint H Ecoscint H Ecoscint H

LS-275 LS-275 LS-275

4 liter (1-3) 4 liter (4+) 20 liter drum

128 183

Nuc-Wipes Nuc-Wipes

NW-300 NW-300

1 box (1-9) (100/box) 1 box (10+) (100/box)

141 202,230

USA: 1-800-526-3867 EUROPE: 441 482 646022

30 31

182

132 189,197

145 200

How to Order - EUROPE Phone: Fax: E-mail:

44 (0) 1482 646020 or 44 (0) 1482 646022 44 (0) 1482 646013 info@agtcbioproducts.com

See page170 for the Index of Subjects

Product Name

Cat. No.

Pack Size

Nuclean 03, 230Nuclean Nuclean

NC-200 NC-200 NC-200

1 quart 1 gallon (1-3) 1 gallon (4+)

Page

Nuclistain Nuclistain

EC-730 EC-730

25 ml (1-3) 25 ml (4 +)

Omnimount Hydromount

HS-110 HS-106

100 ml (1-3) 100 ml (4+)

103 25

OptiClear OptiClear OptiClear OptiClear OptiClear

OE-101 OE-101 OE-101 OE-101 OE-101

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon 55 gallon drum

161 227 227 227 227

OptiClear E OptiClear E OptiClear E OptiClear E

OE-104 OE-104 OE-104 OE-104

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon

162 228 228 228

OptiClear R OptiClear R OptiClear R OptiClear R

OE-102 OE-102 OE-102 OE-102

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon

162 228 228 228

OptiClear S OptiClear S OptiClear S OptiClear S OptiClear S

OE-105 OE-105 OE-105 OE-105 OE-105

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon 55 gallon drum

162 228 228 228 228

OptiClear S2 Opticlear S2 Opticlear S2 Opticlear S2

OE-107 OE-107 OE-107 OE-107

16 oz. spray bottle (1-3) 1 gallon (1-3) 1 gallon (4 +) 5 gallon

163 229 229 229

140 203, 230 203, 230

27 186

Product Name

Cat. No.

Pack Size

SequaGel MD Monomer Solution SequaGel MD Monomer Solution

EC-845 EC-845

200 ml (1-3) 200 ml (4 +)

Page 12 16

SequaGel MD SSCP Kit

EC-846

1 kit

SequaGel MD SSCP Stop Solution

EC-848

1.2 ml

SequaGel XR SequaGel XR SequaGel XR

EC-842 EC-842 EC-842

450 ml 1 liter (1-3) 1 liter (4 +)

12 17 17

SequaGel XR Concentrate SequaGel XR Concentrate SequaGel XR Concentrate

EC-843 EC-843 EC-843

100 ml 450 ml (1-3) 450 ml (4+)

12 17 17

Sodium Acetate 3M pH 4.5

EC-905

200 ml

Sodium Acetate 3M pH 5.2

EC-906

200 ml

Sodium Acetate 3M pH 7.0

EC-907

200 ml

Sodium Chloride 1M

EC-901

1 liter

Sodium Chloride 0.9%

EC-902

1 liter

Soluscint XR Soluscint A

LS-314 LS-313

4 liter (1-3) 4 liter (4+)

Solusol Solusol

LS-311 LS-311

450 ml (1-3) 450 ml (4+)

138 194

SSC Buffer 20X SSC Buffer 20X

EC-873 EC-873

1 liter 4 liters

18 19

SSPE Buffer 20X

EC-910

1 liter

STERLING Rapid Silver Stain STERLING Rapid Silver Stain

EC-720 EC-720

1 kit (1-3) 1 kit (4+)

20 27

TAE Buffer 50X TAE Buffer 50X

EC-872 EC-872

1 liter (1-3) 1 liter (4+)

18 20

TBE 5X TBE 5X TBE 5X

EC-861 EC-861 EC-861

1 liter 4 liters (1-3) 4 liters (4+)

18 20 20

EC-860 EC-860 EC-860

1 liter 4 liters (1-3) 4 liters (4+)

17 139 195

OptiClear Sample Kit I

OE-108

1 liter of each

160

OptiClear Sample Kit II

OE-109

1 liter of each

160

OptiClear W OptiClear W OptiClear W OptiClear W

OE-106 OE-106 OE-106 OE-106

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4 +) 5 gallon

Oxosol 306 Oxosol 306

LS-231 LS-231

1 liter 4 liter

137

TBE 10X TBE 10X TBE 10X

Oxosol C14 Oxidizer Oxosol C14 Oxidizer

LS-211 LS-211

4 liter (1-3) 4 liter (4 +)

TBS (10X)

EC-881

1 liter

17,24

TBST (10X)

EC-882

1 liter

17,24

PBS 10X PBS 10X PBS 10X

CL-253 CL-253 CL-253

450 ml 1 liter 4 liter

17,24 18 18

POPOP POPOP

SFC-60 SFC-60

25 gram 100 gram

145 201

Potassium Acetate, 1M

EC-908

200 ml

Potassium Acetate, 5M

EC-909

200 ml

Potassium Chloride, 1M

EC-903

1 liter

PPO PPO PPO

SFC-10 SFC-10 SFC-10

25 gram 100 gram 500 gram

145 200 200

Protein Loading Buffer Blue 2X

EC-886

10x1 ml

Protein Loading Buffer, 5X

EC-887

10x1 ml

163 229 229 229

137 200

16,26 16

ProtoBlock System

CL-252

1 system

ProtoBlot Transfer Buffer (10X)

EC-878

1 liter

ProtoBlue Safe ProtoGel

EC-722 EC-890

1 liter 4 liter

20 20

ProtoGel (30%) ProtoGel ProtoGel

EC-890 EC-890 EC-890

450 ml 1 liter (1-3) 1 liter (4 +)

6 12 20

ProtoGel (40%) ProtoGel ProtoGel

EC-891 EC-890 EC-890

450 ml 1 liter (1-3) 1 liter (4 +)

6 12 12

ProtoGel Buffer ProtoGel Buffer ProtoGel Buffer

EC-892 EC-892 EC-892

450 ml 1 liter (1-3) 1 liter (4 +)

16,6 12 12

ProtoGel Quick-Cast 12% POPOP

EC-895 SFC-60

100 ml 450 ml

ProtoGel Quick-Cast Loading Buffer

EC-896

5 x 2 ml

ProtoGel Sample Prep Kit

EC-884

1 Kit

24 23

7,16

ProtoGel Stacking Buffer

EC-893

200 ml

16,6

ProtoGlow ECL POPOP

CL-300 SFC-60

200 ml kit 500 ml kit

ProtoLift Western Stripping Buffer

EC-889

100 ml

18 20 20

TE BUFFER 100X

EC-862

25 ml

TEMED

EC-503

25 ml

p-Terphenyl p-Terphenyl

SFC-50 SFC-50

25 gram 100 gram

145

TPB

SFC-15

5 gram

145

Tricine

EC-407

100 gram

Triple Dye Loading Buffer (6X)

EC-855

1.2 ml

Tris Tris

EC-406 EC-406

250 gram 1 kilogram

31 33

Tris-Glycine Electroblotting Buffer 10X Tris-Glycine Electroblotting Buffer 10X Tris-Glycine Electroblotting Buffer 10X

EC-880 EC-880 EC-880

1 liter 4 liter (1-3) 4 liter (4 +)

17,24 21 21

Tris-Glycine-SDS PAGE Buffer (10X) Tris-Glycine-SDS PAGE Buffer (10X) Tris-Glycine-SDS PAGE Buffer (10X)

EC-870 EC-870 EC-870

1 liter 4 liter (1-3) 4 liter (4 +)

16 21 21

Tris-HCl, pH 7.2

EC-922

1 liter

Tris-HCl, pH 7.4

EC-923

1 liter

Tris-HCl, pH 7.6

EC-925

1 liter

Tris-Tricine-SDS PAGE Buffer (10X)

EC-869

1 liter

TTE Glycerol Tolerant Buffer 20X TTE Glycerol Tolerant Buffer 20X

EC-871 EC-871

1 liter (1-3) 1 liter (4 +)

18 21

Tween 20 Tween 20

EC-607 EC-607

200 ml 1 liter

31 188

Uniscint BD Uniscint BD Uniscint BD

LS-276 LS-276 LS-276

4 liter (1-3) 4 liter (4+) 20 liter drum

130, 135 185,193 185,193

Urea Urea

EC-605 EC-605

250 gram 1 kilogram

31 33

UreaGel 29:1 Concentrate UreaGel Concentrate UreaGel Concentrate 15 UreaGel 29:1 System SequaGel Sequencing System SequaGel Sequencing System

EC-828 EC-830 EC-830

450 ml 1 liter (1-3) 1 liter (4 +)

EC-829 EC-833 EC-833

1 liter kit 2.2 liter kit (1-3) 2.2 liter kit (4 +)

10 15

UreaGel-6 UreaGel-6 UreaGel-6

EC-836 EC-836 EC-836

450 ml 1 liter (1-3) 1 liter (4 +)

UreaGel-8 UreaGel-8 UreaGel-8

EC-838 EC-838 EC-838

450 ml 1 liter (1-3) 1 liter (4 +)

10 14 14

UreaGel Buffer UreaGel Buffer UreaGel Buffer

EC-835 EC-835 EC-835

100 ml 200 ml (1-3) 200 ml (4 +)

UreaGel Complete Buffer UreaGel Complete Buffer

EC-841 EC-841

90 ml 200 ml

10 14

UreaGel Concentrate UreaGel Concentrate UreaGel Concentrate

EC-830 EC-830 EC-830

450 ml 1 liter (1-3) 1 liter (4 +)

UreaGel Diluent UreaGel Diluent SequaGel Diluent

EC-840 EC-840 EC-840

450 ml 1 liter (1-3) 1 liter (4 +)

11 15 15

UreaGel Loading Buffer

EC-857

10 x 1ml

EC-833 EC-833 EC-833

1 liter kit 2.2 liter kit (1-3) 2.2 liter kit (4 +)

11 15

10 15

10 14 14

ProtoMarkers

EC-898

0.5 ml

ProtoMetrics

EC-899

1 Tube

ProtoStain Blue

EC-727

1 liter

Pyronin Y

HS-607

5 gram

105

Riboflavin

EC-501

25 gram

Scintillation Vials 6 mL Scintillation Vials 6 mL

SVC-06 SVC-06

1 case (1-10) 1 case (11+)

142 203

Scintillation Vials 8 mL Scintillation Vials 6 mL

SVC-08 SVC-06

1 case (1-10) 1 case (11+)

142 203

Scintillation Vials 20 mL Scintillation Vials 20 mL

SVC-20 SVC-20

1 case (1-10) 1 case (11+)

142

Scottâ&#x20AC;&#x2122;s Tapwater

HS-404

1 liter

104

SDS SDS

EC-604 EC-604

100 gram 1 kilogram

31 33

SDS Solution 20% SDS Solution 20%

EC-874 EC-874

100 ml 450 ml

31 19

UreaGel Sequencing System SequaGel Sequencing System SequaGel Sequencing System

SequaGel MD Heteroduplex Kit

EC-847

1 kit

Water, DEPC treated, sterile

EC-625

1 liter

Xylene Cyanole FF

HS-608

25 grams

19 31

USA: 1-800-526-3867 EUROPE: 441 482 646022

11 15 15

Index of Products

11 15 15

167 761

How to Order - USA Toll - Free Phone: 800-526-3867 Telephone: 404-699-2121 Order by Fax: 404-699-2077 Order on the Web: www.nationaldiagnostics.com E-mail: info@nationaldiagnostics.com

Numerical Product Index Cat. No.

Product Name

Pack Size

CL-202

Diogenes

1 kit

Index of Products

Product Name

EC-840 UreaGel Diluent UreaGel Diluent EC-840 UreaGel Diluent EC-840

Pack Size

Page 11 15 15

EC-841 UreaGel Complete Buffer SequaGel Complete Buffer EC-841

90 ml 200 ml

10 14

22 31

EC-842 SequaGel XR SequaGel XR EC-842 SequaGel XR EC-842

450 ml 1 liter (1-3) 1 liter (4 +)

12 17 17

30 30 30

EC-843 SequaGel XR Concentrate SequaGel XR Concentrate EC-843 SequaGel XR Concentrate EC-843

100 ml 450 ml (1-3) 450 ml (4 +)

12 17 17

EC-845 SequaGel MD Monomer Solution SequaGel MD Monomer Solution EC-845

200 ml (1-3) 200 ml (4 +)

Hydrogen Peroxide Assay Kit

100 Assay kit

ProtoBlock System

1 system

CL-253 PBS 10X PBS 10X

PBS 10X CL-253 CL-253

450 ml 1 liter 4 liters

17,24 18 18

CL-300 ProtoGlow ECL Dithiothreitol (DTT) EC-601

200 ml system 500 ml system

EC-201 Acrylamide Acrylamide EC-201 Acrylamide EC-201

100 grams 500 grams 1 kilogram

EC-202 AquaPor LE AquaPor LE EC-202 AquaPor LE EC-202 AquaPor LE EC-202

25 grams 100 grams (1-3) 100 grams (4+) 500 grams

14 8 8 8

EC-203 AquaPor ES AquaPor ES EC-203 AquaPor ES EC-203

25 grams 100 grams (1-3) 100 grams (4+)

15 9 9

EC-204 AquaPor LM AquaPor LM EC-204 AquaPor LM EC-204

25 grams 100 grams (1-3) 100 grams (4+)

14 8 8

EC-205 AquaPor HR AquaPor HR EC-205 AquaPor HR EC-205

25 grams 100 grams (1-3) 100 grams (4+)

EC-206 AquaPor 3:1 AquaPor ES EC-203 AquaPor ES EC-203

25 grams 100 grams (1-3) 100 grams (4 +)

15 9 9

EC-301

Bis (Methylene Bis-Acrylamide)

25 grams

EC-303

DATD

25 grams

EC-405 Glycine Glycine EC-405

250 grams 1 kilogram

31 32

EC-406 Tris Tris EC-406

250 grams 1 kilogram

31 33

EC-407

Tricine

100 grams

EC-408

Ion Exchange Resin

100 grams

EC-501

Riboflavin

25 grams

EC-503

TEMED

121

15 9 9

25 ml

EC-504 Ammonium Persulfate Ammonium Persulfate EC-504

25 grams 100 grams

30 30

EC-601 Dithiothreitol (DTT) Dithiothreitol (DTT) EC-601

1 gram 5 grams

30 31

EC-603

50 ml

EC-604 SDS SDS EC-604

100 grams 1 kilogram

31 33

EC-605 Urea Urea EC-605

250 grams 1 kilogram

31 33

EC-606

Mercaptoethanol

Cat. No.

450 ml 1 liter (1-3) 1 liter (4 +)

CL-204 CL-252

12 16

EC-846

SequaGel MD SSCP Kit

1 kit

EC-847

SequaGel MD Heteroduplex Kit

1 kit

EC-848

SequaGel MD SSCP Stop Solution

1.2 ml

EC-849 AccuGel 19:1 (30%) AccuGel 19:1 (40%) EC-850 AccuGel 19:1 (40%) EC-850

450 ml 1 1iter (1-3) 1 liter (4+)

13 6 6

EC-850 AccuGel 19:1 (40%) AccuGel 19:1 (40%) EC-850 AccuGel 19:1 (40%) EC-850

450 ml 1 1iter (1-3) 1 liter (4+)

13 6 6

EC-851 AccuGel 29:1 (30%) AccuGel 19:1 (40%) EC-850 AccuGel 19:1 (40%) EC-850

450 ml 1 1iter (1-3) 1 liter (4+)

8, 13 6 6

EC-852 AccuGel 29:1 (40%) AccuGel 29:1 (40%) EC-852 AccuGel 29:1 (40%) EC-852

450 ml 1 liter (1-3) 1 liter (4+)

8, 13 6 6

EC-855

Triple Dye Loading Buffer (6X)

1.2 ml

EC-857

UreaGel Loading Buffer

10 x 1ml

EC-860 TBE 10X TBE 10X

TBE 10X EC-860 EC-860

1 liter 4 liters (1-3) 4 liters (4+)

18 20 20

EC-861 TBE 5X TBE 5X

TBE 5X EC-861 EC-861

1 liter 4 liters (1-3) 4 liters (4+)

18 20 20

EC-862

TE BUFFER 100X

25 ml

EC-867 MOPS-SDS Running Buffer (20X) Dextran Sulfate EC-877

450 ml 1 liter

16 31

EC-868 MES-SDS Running Buffer Dextran Sulfate EC-877

450 ml 1 liter

16 31

EC-869

1 liter

1 liter 4 liter (1-3) 4 liter (4+)

16,23 21 21

EC-871 TTE Glycerol Tolerant Buffer (20X) TTE Glycerol Tolerant Buffer 20X EC-871

1 liter (1-3) 1 liter (4+)

18 21

EC-872 TAE Buffer 50X TAE Buffer 50X EC-872

1 liter (1-3) 1 liter (4+)

18 20

Tris-Tricine-SDS PAGE Buffer (10X)

EC-870 Tris-Glycine-SDS PAGE Buffer (10X) Tris-Glycine-SDS PAGE Buffer (10X) EC-870 Tris-Glycine-SDS PAGE Buffer (10X) EC-870

Glycerol

450 ml

EC-607 Tween 20 Tween 20 EC-607

200 ml 1 liter

EC-608 Formamide

200 ml 1 gallon

EC-873 SSC Buffer 20X SSC Buffer 20X EC-873

1 liter 4 liters

18 19

EC-609

EC-874 SDS Solution 20% SDS Solution 20% EC-874

100 ml 450 ml

31 19

EC-875 Denaturation Solution Denaturation Solution EC-875

1 liter (1-3) 1 liter (4+)

EC-876 Neutralization Solution Neutralization Solution EC-876

1 liter (1-3) 1 liter (4+)

19 18

31 33

Boric Acid

500 grams

EC-610 EDTA EDTA EC-610

100 grams 500 grams

30 31

EC-612

GelDry Film 11 X 12 cm

50 sheets

EC-620

Glass Bond

25 ml

EC-621

Glass Free

450 ml

19 18

EC-622

GelDry Film 22.5 X 22.5 cm

50 sheets

EC-877 Dextran Sulfate Dextran Sulfate EC-877

50 grams 250 grams

EC-625

Water, DEPC treated sterile

1 liter

EC-878

1 liter

17,23

EC-720 STERLING Rapid Silver Stain STERLING Rapid Silver Stain EC-720

1 kit (1-3) 1 kit (4+)

20 27

EC-722 ProtoBlue Safe STERLING Rapid Silver Stain EC-720

450 ml 1 liter 4 liter

EC-880 Tris-Glycine Electroblotting Buffer (10X) Tris-Glycine Electroblotting Buffer 10X EC-880 Tris-Glycine Electroblotting Buffer 10X EC-880

1 liter 4 liter (1-3) 4 liter (4+)

17,24 21 21

EC-881

TBS (10X)

1 liter

EC-882

TBST(10X)

1 liter

17,24 26

ProtoBlot Transfer Buffer (10X)

30 31

17,24

1 liter

EC-884

ProtoGel Sample Prep Kit

1 Kit

EC-730 Nuclistain Nuclistain EC-730

25 ml (1-3) 25 ml (4+)

27 25

EC-886

Protein Loading Buffer Blue 2X

10x1 ml

EC-810 AcrylaGel AcrylaGel EC-810 AcrylaGel EC-810

450 ml 1 liter (1-3) 1 liter (4 +)

9,13 7 7

EC-887

5X Protein Loading Buffer

10x1 ml

EC-888

ND Protein Precipitation Kit

1 kit

EC-820 Bis-AcrylaGel Bis-AcrylaGel EC-820 Bis-AcrylaGel EC-820

450 ml 1 liter (1-3) 1 liter (4 +)

9,13 7 7

EC-889

ProtoLift Western Stripping Buffer

100 ml

EC-828 UreaGel 29:1 Concentrate UreaGel Concentrate EC-830 UreaGel Concentrate EC-830

450 ml 1 liter (1-3) 1 liter (4 +)

EC-890 ProtoGel (30%) ProtoGel EC-890 ProtoGel EC-890

450 ml 1 liter (1-3) 1 liter (4 +)

6 12 12

EC-891 ProtoGel (40%) ProtoGel EC-890 ProtoGel EC-890

450 ml 1 liter (1-3) 1 liter (4 +)

6 12 12

EC-829 UreaGel 29:1 Sequencing System UreaGel Sequencing System EC-833 UreaGel Sequencing System EC-833

1 liter kit 2.2 liter kit (1-3) 2.2 liter kit (4 +)

10 15 15

EC-892 ProtoGel Buffer ProtoGel Buffer EC-892 ProtoGel Buffer EC-892

450 ml 1 liter (1-3) 1 liter (4 +)

16,6 12 12

EC-830 UreaGel Concentrate UreaGel Concentrate EC-830 UreaGel Concentrate EC-830

450 ml 1 liter (1-3) 1 liter (4 +)

200 ml

16,6

EC-833 UreaGel Sequencing System UreaGel Sequencing System EC-833 UreaGel Sequencing System EC-833

1 liter kit 2.2 liter kit (1-3) 2.2 liter kit (4 +)

11 15 15

EC-895 ProtoGel Quick-Cast 12% ProtoGel Buffer EC-892

100 ml 450 ml

7 12

EC-896

ProtoGel Quick-Cast Loading Buffer

5x1 ml

7,16

EC-835 UreaGel Buffer UreaGel Buffer EC-835 UreaGel Buffer EC-835

100 ml 200 ml (1-3) 200 ml (4 +)

EC-836 UreaGel-6 UreaGel-6 EC-836 UreaGel-6 EC-836

450 ml 1 liter (1-3) 1 liter (4 +)

EC-838 UreaGel-8 UreaGel-8 EC-838 UreaGel-8 EC-838

450 ml 1 liter (1-3) 1 liter (4 +)

EC-727

168

Page 120

ProtoStain Blue

USA: 1-800-526-3867 EUROPE: 441 482 646022

10 15

11 15 15

EC-893

ProtoGel Stacking Buffer

16,26 26

EC-897

Insite Markers

1 Tube

EC-898

ProtoMarkers

0.5 ml

EC-899

ProtoMetrics

1 Tube

10 14 14

EC-900

EDTA, 0.5M Solution

1 liter

EC-901

Sodium Chloride, 1M

1 liter

10 14

EC-902

Sodium Chloride, 0.9%

1 liter

EC-903

Potassium Chloride

1 liter

11 15 15

How to Order - EUROPE Phone: Fax: E-mail: Cat. No.

44 (0) 1482 646020 or 44 (0) 1482 646022 44 (0) 1482 646013 salesuk@nationaldiagnostics.com

Product Name

See page 170 for the Index of Subjects

Pack Size

Page

EC-905

Sodium Acetate, pH 4.5

1 liter

EC-906

Sodium Acetate, pH 5.2

1 liter

EC-907

Sodium Acetate, pH 7.0

1 liter

Cat. No.

Product Name

Pack Size

Page

EC-908

Potassium Acetate, 1M

1 liter

EC-909

Potassium Acetate, 5M

1 liter

EC-910

SSPE (20X)

1 liter

EC-911

MESA RNA Electrophoresis Buffer (10X)

1 liter

EC-915

Denhardt’s Solution

50 ml

EC-922

Tris-HCl, pH 7.2

1 liter

EC-923

Tris-HCl, pH 7.4

1 liter

EC-925

Tris-HCl, pH 7.6

LS-274 Ecoscint O Ecoscint O LS-274 Ecoscint O LS-274

4 liter (1-3) 4 liter (4+) 20 liter drum

129 184 184

LS-275 Ecoscint H Ecoscint H LS-275 Ecoscint H LS-275

4 liter (1-3) 4 liter (4+) 20 liter drum

128 183

LS-276 Uniscint BD Uniscint BDLS-276 Uniscint BDLS-276

4 liter (1-3) 4 liter (4+) 20 liter drum

130,135 185,193 185,193

LS-281 Monoflow 1 Monoflow 1 LS-281 Monoflow 1 LS-281

4 liter (1-3) 4 liter (4+) 20 liter drum

LS-282 Monoflow 2 Monoflow 2 LS-282 Monoflow 2 LS-282

4 liter (1-3) 4 liter (4+) 20 liter drum

136 192 192

LS-283 Monoflow 3 Monoflow 3 LS-283 Monoflow 3 LS-283

1x4 liter bottle 4x4 liter case 20 liter drum

136 192 192

LS-284 Monoflow 4 Monoflow 4 LS-284 Monoflow 4 LS-284

4 liter (1-3) 4 liter (4+) 20 liter drum

136 193 193

136 192 192

1 liter

HS-100 Histosol Histosol H -100

1 gallon 5 gallon

99 149

HS-101 Mirsky’s Fixative (ready-to-use) Mirsky’s Fixative (ready-to-use) HS-101

1 gallon 5 gallon

HS-102 Mirsky’s Fixative Mirsky’s Fixative HS-102

200 ml system 2 liter system

101 151

HS-103 Histomount Histomount HS-103 Histomount HS-103 Histomount HS-103

100 ml (1-3) 100 ml (4+) 450 ml (1-3) 450 ml (4+)

103 153 153 153

LS-285 Monoflow 5 Monoflow 5 LS-285 Monoflow 5 LS-285

4 liter (1-3) 4 liter (4+) 20 liter drum

135 193 193

HS-104 Calci-Clear Calci-Clear HS-104 Calci-Clear HS-104

1 quart 1 gallon 5 gallons

100 150 150

LS-288 Ecoscint Flow Ecoscint A LS-273 Ecoscint A LS-273

4 liter (1-3) 4 liter (4+) 4 liter (4+)

HS-105 Calci-Clear Rapid Calci-Clear Rapid HS-105 Calci-Clear Rapid HS-105

1 quart 1 gallon 5 gallons

100 150 150

LS-309 Bioscint Bioscint LS-309

4 liter (1-3) 4 liter (4+)

138 194

HS-106 Hydromount Hydromount HS-106

100 ml (1-3) 100 ml (4+)

103 153

400 ml

138

HS-108

Neutralin

5 gallon pail

102

LS-311 Solusol Solusol LS-311

450 ml (1-3) 450 ml (4+)

138 194

HS-110 Omnimount Hydromount HS-106

100 ml (1-3) 100 ml (4+)

103 153

LS-314 Soluscint XR Soluscint O LS-312

1 gallon (1-3) 1 gallon (4+)

139 195

HS-200 Histo-Clear Histo-Clear HS-200 Histo-Clear HS-200

1 liter 1 gallon (1-3) 1 gallon (4+) 5 gallon 55 gallon

98 148 148

LS-315 Autofluor

Autofluor LS-315

1 liter (1-3) 1 liter (4+)

28,144 153

LS-900

Bottle-top Dispenser

1 unit

143

LS-904

Extendable Delivery Jet

1 unit

143

HS-202 Histo-Clear II Histo-Clear II HS-202 Histo-Clear II HS-202

1 gallon (1-3) 1 gallon (4+) 5 gallon 55 gallon

99 149 149

NC-200 Nuclean Nuclean NC-200 Nuclean NC-200

1 quart 1 gallon (1-3) 1 gallon (4+)

NW-300 Nuc-Wipes Nuc-Wipes NW-300

1 box (1-9) (100/box) 1 box (10+) (100/box)

141 202,230

HS-300 Reagent Ethanol - Denatured Liquiscint LS-121 Liquiscint LS-121

1 liter 4 liter 20 liter drum

30,99 190 188

OE-101 OptiClear OptiClear OE-101 OptiClear OE-101 OptiClear OE-101 OptiClear OE-101

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon 55 gallon drum

OE-102 OptiClear R OptiClear R OE-102 OptiClear R OE-102 OptiClear R OE-102

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon drum

OE-104 OptiClear E OptiClear E OE-104 OptiClear E OE-104 OptiClear E OE-104

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon drum

228

OE-105 OptiClear S OptiClear S OE-105 OptiClear S OE-105 OptiClear S OE-105 OptiClear S OE-105

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon drum 55 gallon drum

162 228 228 228 228

OE-106 OptiClear W OptiClear W OE-106 OptiClear W OE-106 OptiClear W OE-106

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon drum

OE-107 OptiClear S2 Opticlear S2 OE-107 Opticlear S2 OE-107 Opticlear S2 OE-107

16 oz. spray bottle 1 gallon (1-3) 1 gallon (4+) 5 gallon drum

163 229 229 229

OE-108

OptiClear Sample Kit I

1 liter of each

160

OE-109

OptiClear Sample Kit II

1 liter of each

160

25 grams 100 grams 500 grams

145 200 200

101 151

HS-400

Harris’ Hematoxylin

1 liter

104

HS-402

Eosin, 1% Solution

100ml

104

HS-404

Scott’s Tapwater

1 liter

104

HS-504

Alcian Blue

25 grams

105

HS-506

Biebrich Scarlet

25 grams

105

HS-516

Fast Green FCF

25 grams

105

HS-518

Basic Fuchsin

25 grams

HS-525

Methylene Blue

25 grams

105

HS-601

Amido Black 10B

25 grams

21,105

HS-602

Bromocresol Green

5 grams

HS-603

Bromophenol Blue

10 grams

21,28,105

HS-604

Coomassie Blue R-250

10 grams

105

HS-605

Coomassie Blue G-250

10 grams

HS-606

Methyl Green

10 grams

105

HS-607

Pyronin Y

5 grams

105

HS-608

Xylene Cyanole FF

25 grams

LS-111 Hydrofluor Hydrofluor LS-111

4 liter (1-3) 4 liter (4 +)

131 186

LS-121 Liquiscint Liquiscint LS-121 Liquiscint LS-121

4 liter (1-3) 4 liter (4 +) 20 liter drum

133 190 188

LS-151 Betafluor

Betafluor LS-151

4 liter (1-3) 4 liter (4 +)

133 191

LS-191 Monofluor Monofluor LS-191

4 liter (1-3) 4 liter (4 +)

132 189,197

LS-201 Filtron-X Filtron-X LS-201

4 liter (1-3) 4 liter (4 +)

139 195

LS-211 Oxosol C14 Oxidizer Oxosol C14 Oxidizer211

4 liter (1-3) 4 liter (4 +)

137 200

LS-231 Oxosol 306 Oxosol 306 LS-231

1 liter 1 gallon

137

LS-241 Carbamate-1 CO2 Absorber Carbamate-1 CO2 Absorber

450 ml (1-3) 450 ml (4 +)

137 196

LS-262

Ecoscint GL

130

4 liter (1-3) 4 liter (4+)

126 184

LS-271 Ecoscint Ecoscint LS-271 Ecoscint LS-271

4 liter (1-3) 4 liter (4+) 20 liter drum

129 184 184

LS-272 Ecoscint XR Ecoscint A LS-273

4 liter (1-3) 4 liter (4+)

127 182

LS-273 Ecoscint A Ecoscint A LS-273 Ecoscint A LS-273

4 liter (1-3) 4 liter (4+) 20 liter drum

128 182

Biosol

SFC-10 PPO PPO SFC-10 PPO SFC-10

182

140 203, 230 203, 230

161 227 227 227 227

162 228 228 228 162 228 228

163 229 229 229

SFC-13 BBQ

BBQ SFC-13

1 gram 5 grams

145 201

SFC-15

TPB

5 grams

145

25 grams 100 grams 500 grams

145 200 200

SFC-20 Butyl PBD Butyl PBD SFC-20 Butyl PBD SFC-20 SFC-40 Naphthalene — Scintillation Grade Naphthalene — Scintillation Grade SFC-40

100 grams 1 kilogram

SFC-50 p-Terphenyl p-Terphenyl SFC-50

25 g 100 g

145

SFC-60 POPOP POPOP SFC-60

25 grams 100 grams

145 201

145 200

SFC-90 Bis-MSB Bis-MSB SFC-90

25 grams 100 grams

145 201

SVC-06 Scintillation Vials 6 mL Scintillation Vials 20 mL SVC-20

1 case (1-10) 1 case (11 +)

142 203

SVC-08 Scintillation Vials 8 mL Scintillation Vials 6 mL SVC-06

1 case (1-10) 1 case (11 +)

142 203

SVC-20 Scintillation Vials 20 mL Scintillation Vials 6 mL SVC-06

1 case (1-10) 1 case (11 +)

142

USA: 1-800-526-3867 EUROPE: 441 482 646022

Index of Products

4 liter

LS-270 Ecoscint Ultra Ecoscint LS-271

LS-310

134

169 961

Subject Index B

A260 of DNA samples.......................................................... 43 AccuGel 19:1...........................................................................13 AccuGel 29:1...................................................................... 8,13 AcrylaGel............................................................................. 9,13 Acrylamide........................................................................ 30,36 Agarose........................................................................ 14,36,37 extra strength...................................................................................60 AquaPor ES.................................................................................. 15 low melting point.............................................................................60 AquaPor LM................................................................................. 14

Alcohol Fixatives.............................................................108,111 Aldehyde fixatives................................................................ 107

disposal with Neutralin........................................................ 102,108 formaldehyde......................................................................... 107,111 formalin-heme pigment..........................................................108,111 gluteraldehyde...............................................................................117 Mirskyâ&#x20AC;&#x2122;s fixative......................................................................107,117

Alkaline blotting.......................................................................81 Alkaline phosphatase...................................................... 88,116 Alpha particles...................................................................... 147 ionization detection.......................................................................148 penetration.....................................................................................147 solid scintillation............................................................................148

Amido Black.............................................................................21 Amino acid oxidase.................................................................75 Ammonium persulfate............................................................. 36 Ammonium Persulfate............................................................. 30 Ampflified Fragment Length Polymorphism (AFLP*)............ 48 Ampholytes.............................................................................. 77 Antibodies.........................................................................76,115 detection systems direct............................................................................................115 indirect.........................................................................................115 production.......................................................................................115

Antigen................................................................................... 115 AquaPor GTAC Agarose.........................................................14 AquaPor 3:1.................................................................................... 15 AquaPor ES...................................................................................... 15 AquaPor HR..................................................................................... 15 AquaPor LE...................................................................................... 14 AquaPor LM..................................................................................... 14

Artifacts in histologic sections................................................111 Autofluor.................................................................................. 28 protocol............................................................................................83

AutoFluor............................................................................... 144 Automated Sequencing.......................................................... 47

Index of Subjects

electrophoresis.................................................................................47 reactions...........................................................................................47 SequaGel XR................................................................................... 12

Autoradiography............................................................ 83,155 Autofluor..........................................................................................28 protocol.........................................................................................83 detection limits.................................................................................83

Avidin-Biotin system............................................................... 116

170

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Base pairs................................................................................ 33 Becquerel.............................................................................. 147 Betafluor.................................................................................133 Beta particles........................................................................ 147

ionization detection.......................................................................148 liquid scintillation detection..................................................148,149 efficiency.....................................................................................150 neutrino emission...........................................................................147 path length.....................................................................................149 secondary emissions.....................................................................147 solid scintillation............................................................................148

Biosol and Bioscint................................................................ 138 Biotin .................................................................................... 116 Bis-AcrylaGel....................................................................... 9,13 Blotting membranes.................................................................81 nitrocellulose.................................................................................... 81 nylon ................................................................................................ 81 PVDF ................................................................................................89

Boric Acid................................................................................ 30 BromoChloroIndoyl Phosphate (BCIP).................................. 88 Bromocresol Green................................................................. 28 Bromophenol Blue............................................................. 21,28 Buffer additives....................................................................... 40 Buffer gradient gels........................................................... 44,46 protocol............................................................................................47

Buffers ............................................................................... 38,44 discontinuous...................................................................................39 homogeneous..................................................................................39 pre-mixed electrophoresis buffers.................................................. 16 selection parameters.......................................................................38 C

Calci-Clear............................................................................100 Calci-Clear Rapid.................................................................100 Calcium deposits...................................................................109 Capillary blotting.................................................................... 80 Carbamate-1.........................................................................137 Carbon Dioxide counting..................................................... 156 Catalase...................................................................................75 Cellulose-Ester Filters............................................................ 155 Channels ratio........................................................................151 Charge to mass ratio............................................. 33,39,54,67 Chemiluminescence.............................................................. 152 Chromophore......................................................................... 112 Clearing.................................................................................109 safer clearing agents Histo-Clear...................................................................................98 Histo-Clear II................................................................................99 Histosol.........................................................................................99

Coincidence counting.......................................................... 152 Color perception.....................................................................111 Conformational analysis........................................................ 58 heteroduplex analysis.....................................................................58 protocol.........................................................................................58 single strand conformational polymorphism (SSCP) a.................59

continued

protocol.........................................................................................59

Coomassie Blue G-250..........................................................21 Coomassie Blue R-250...........................................................21 Coomassie staining................................................................. 85 powdered stains.............................................................................. 21

Counting efficiency............................................................... 150 Counting Efficiency............................................................... 150 Counting windows................................................................ 150 Counts per minute..........................................................147,150 Curie 147

DNA and RNA Purification

electroelution.............................................................................62,63 using agarose..................................................................................62 using low melt agarose...................................................................62

DNA footprint analysis............................................................51 DNA/Protein interactions.......................................................51 DNase I footprinting................................................................51 standard protocol............................................................................ 51

DNA sequencing

electrophoresis.................................................................................46 G-C compressions.......................................................................47 glycerol.........................................................................................47 gradient gels.................................................................................47 TTE buffer......................................................................................47 Maxam and Gilbert........................................................................45 Sanger dideoxy terminators...........................................................45

DATD 30 Decalcification......................................................................109 Calci-Clear and CalciClear Rapid............................................. 100

Decolorizing LSC samples................................................... 154 Decolorizing LSC Samples with Ultraviolet Light protocols........................................................................................154

Dehydration.....................................................................109,111 dioxane......................................................................................... 109

Denaturing agents............................................................ 43,44 DEPC (Diethylpyrocarbamate).............................................. 65 Dextran Sulfate....................................................................... 30 Diaminobenzidine......................................................75,88,116 Differential display.................................................................. 48 Diogenes............................................................................... 120 Dioxane.................................................................................109 Discontinuous buffer systems............................................39,67 Disintegrations per minute.................................................... 150 Disintegrations per minute (DPM)........................................ 147 Dithiothreitol...................................................................... 40,67 Dithiothreitol (DTT).................................................................. 30 DNA and RNA Detection....................................................... 27 autoradiography.............................................................................28 ethidium bromide.............................................................................79 protocol.........................................................................................79 nuclistain..........................................................................................27 protocol.........................................................................................79 silver staining protocol.........................................................................................80 Southern blotting.............................................................................80 protocol.........................................................................................82

DNA and RNA Electrophoresis............................................. 42

Dyes .....................................................................................111 Chromophores................................................................................112

Ecoscint...................................................................................129 Ecoscint A...............................................................................128 Ecoscint Flow..........................................................................134 Ecoscint H...............................................................................128 Ecoscint O..............................................................................129 Ecoscint XR.............................................................................127 EDTA ..................................................................................... 30 Ehrlichâ&#x20AC;&#x2122;s Hematoxylin............................................................ 113 Electroendosmosis.............................................................37,60 Electron microscopy............................................................... 116 fixation.............................................................................................117 immunostaining...............................................................................117 resolution.........................................................................................116 scanning electron microscope.......................................................116 sectioning........................................................................................117 staining............................................................................................117 negative stains.............................................................................117 positive stains..............................................................................117 tissue processing.............................................................................117 transmission electron microscope..................................................116

Electro-optical solvents......................................................... 160 Electrophoresis reagents......................................................... 30 Embedding.............................................................................110 electron microscopy.......................................................................117 Epoxy resins................................................................................... 110 media............................................................................................. 110 Paraffin wax................................................................................... 110

Enzyme linked immunosorbent assay................................... 89 Epitope.................................................................................... 115 Epoxy resins...........................................................................110 Ethidium bromide...............................................................43,79 F

Ferguson plots..........................................................................74 Field inversion gel electrophoresis (FIGE)............................. 64 Filtron X.................................................................................. 139 Fixation (histology)....................................................... 107,108 alcohols......................................................................................... 108 aldehydes...............................................................................107,117 Mirskyâ&#x20AC;&#x2122;s Fixative.........................................................................101 USA: 1-800-526-3867 EUROPE: 441 482 646022

Index of Subjects

agarose gel electrophoresis...........................................................60 AquaPor GTAC agarose............................................................. 14 gel preparation............................................................................60 gel preparation - alkaline gels.................................................... 61 gel preparation - formaldehyde gels......................................... 61 troubleshooting.............................................................................93 denaturing electrophoresis of DNA & RNA...................... 33,40,43 gel preparation............................................................................43 manual sequencing......................................................................44 molecular weight determination.................................................44 run conditions...............................................................................44 troubleshooting.............................................................................92 gels for DNA and RNA analysis....................................................10 native DNA electrophoresis............................................................54 gel formulations............................................................................54 sample preparation......................................................................54

continued

pre-mixed electrophoresis buffers.................................................. 18

171 171

Alphabetical Index F continued Aldehydes......................................................................................107 electron microscopy.......................................................................116 glutaraldehyde............................................................................117 Mirskyâ&#x20AC;&#x2122;s fixative...................................................................101,117 osmium tetroxide.........................................................................117 mercurials........................................................................ 107,108,111 oxidizing agents................................................................... 107,108 pH ............................................................................................. 108 picric acid..................................................................................... 108 safety ............................................................................................. 108 table of fixatives............................................................................107 temperature................................................................................... 108

Fixation of protein gels........................................................... 84 protocol............................................................................................84

Flow counting........................................................................ 156 applications table..........................................................................156 biodegradable cocktails for flow detection................................134 detectors.........................................................................................156 traditional cocktails for flow detection.........................................136

Flow detectors....................................................................... 156 Fluorography................................................................... 83,155 detection limits.................................................................................83 protocol............................................................................................83 TLC plates protocol.......................................................................................155

Formaldehyde.................................................................107,111 Formaldehyde gels................................................................. 65 Formalin-heme pigment..................................................108,111 Formamide....................................................................30,43,44 Frozen sections.......................................................................110 G

ionization detection.......................................................................148

Hematoxylin and Eosin stain................................................. 113 Henderson-Hasselbalch equation......................................... 38 H & E Stain............................................................................. 113 Heteroduplex analysis............................................................ 58 SequaGel MD................................................................................. 12

Histo-Clear.............................................................................. 98 Histo-Clear II........................................................................... 99 Histological artifacts...............................................................111 Histological Stains................................................................ 104 Histomount.............................................................................103 Histosol.................................................................................... 99 Homogeneous buffer systems................................................ 39 Horizontal gels.........................................................................41 Horseradish peroxidase...................................... 75,88,115,116 Human eye..............................................................................111 Hydrofluor..............................................................................131 Hydrogen bonding agents..................................................... 40 Hydromount..........................................................................103 I protocol............................................................................................76

Immuno-electrophoresis..........................................................76 protocol............................................................................................76

Gamma rays

Secondary b emissions................................................................147

G-C compressions.................................................................. 47 GelDry Film............................................................................. 29 Gel preparation agarose gels....................................................................................60 denaturing DNA gels......................................................................43 denaturing protein gels...................................................................68 gradient gels.................................................................................69 native DNA gels..............................................................................54 native protein gels........................................................................... 75

Gels for electrophoresis

Index of Subjects

continued

Immuno-diffusion.....................................................................76

Gamma rays.......................................................................... 147

gels for DNA/RNA analysis..........................................................10 AccuGel 19:1............................................................................... 13 AcrylaGel and Bis-AcrylaGel.................................................... 13 SequaGel 4, 4.25, 4.75, 6, and 8...........................................10 SequaGel MD.............................................................................. 12 SequaGel XR................................................................................ 12 gels for protein analysis.................................................................... 6 AccuGel 19:1............................................................................... 13 AccuGel 29:1................................................................................ 8 AcrylaGel and BisAcrylaGel........................................................ 9 ProtoGel.......................................................................................6,7

Genomic Analysis................................................................... 48 Ampflified Fragment Length Polymorphism (AFLP*).....................48 Random Amplification of Polymorphic DNA (RAPD)...................48

Glass Bond.............................................................................. 29 172

Glass Free................................................................................ 29 Gluteraldehyde...................................................................... 117 Glycerol.............................................................................. 31,44 Glycine.....................................................................................31 Glyoxal gels for RNA............................................................. 65 Gradient gels.....................................................................69,70 Guanidinium isothiocyanate.................................................. 65 Guide strip Technique............................................................. 87

USA: 1-800-526-3867 EUROPE: 441 482 646022

Immunohistochemistry........................................................... 115 Avidin-biotin system.......................................................................116 detection systems............................................................................115 Enzyme-antibody conjugates........................................................116 immunofluorescence......................................................................115 peroxidase-antiperoxidase...........................................................116

Immunological detection (electrophoresis)......................87,88 In - gel reactions ligation.............................................................................................63 restriction digestion..........................................................................63

Insite Markers.......................................................................... 25 Ionization detection.............................................................. 148 Isoelectric focusing............................................................34,77 protocol............................................................................................77

Isoelectric point..................................................................34,77 Isotachophoresis..................................................................... 40 Isotopes................................................................................. 147

decay products (table)..................................................................147 detection and quantitation............................................................147 energy spectrum.................................................................... 150,151 half life............................................................................................147 K

Kohlrausch discontinuity....................................................39,67 L

Laemmli gel system.....................................................6,7,39,67

continued

Leading ions............................................................................ 39 Ligation, in - gel...................................................................... 63 Limiting mobility...................................................................... 64 Liquid scintillation.................................................................. 148 Liquid scintillation cocktails...................................................124 biodegradable scintillation cocktails...........................................126 choosing a scintillation fluid.........................................................125 cocktails for flow detection...........................................................134 sample oxidation solutions...........................................................137 tissue/gel/filter solubilization......................................................138 traditional scintillation cocktails.................................................... 131

Liquid scintillation counting

alkaline samples............................................................................154 beta particles.................................................................................148 Carbon dioxide.....................................................................155,156 protocol.......................................................................................156 sample oxidation solutions........................................................137 Cellulose-Ester Filters....................................................................155 chemiluminescence............................................................... 152,154 cocktails......................................................................................... 151 biodegradable................................................................... 126,152 sample capacity.........................................................................152 coincidence counting....................................................................152 Counting efficiency.......................................................................150 Counting windows.........................................................................150 Decolorizing..................................................................................154 HPLC flow counting.......................................................................156 mechanism.....................................................................................148 phosphors...............................................................................145,149 Polyacrylamide gels protocol.......................................................................................156 quenching......................................................................................150 Radiation safety wipe testing.................................................................................157 signal interpretation.......................................................................149 light emission patterns................................................................149 pulse analysis.............................................................................150 solvents...........................................................................................148 static electricity..............................................................................152 Tissue samples...............................................................................155 protocol.......................................................................................155 troubleshooting..............................................................................158 waste disposal...............................................................................152

continued

Mercurial fixatives..........................................................108,111 Methylation interference...................................................51,53 Methylenebisacrylamide....................................................... 36 Methylene Bisacrylamide...................................................... 30 Microtomy.............................................................................. 117 Microwave fixation- Mirsky’s fixative................................. 107 protocol..........................................................................................107

Mini-gels...................................................................................41 Mirsky’s fixative............................................................. 107,117 microwave enhancement..............................................................107

Mirsky’s Fixative.................................................................... 101 Mobility shift assay................................................................. 57 protocol............................................................................................57

Monoflow 1 - 4.................................................................... 136 Monoflow 5.......................................................................... 135 Monofluor..............................................................................132 Mounting of tissues..........................................................111,113 mounting media............................................................................ 103 Histomount................................................................................. 103 Hydromount............................................................................... 103 protocol...........................................................................................113 N

Napthalene........................................................................... 149 Neutralin........................................................................102,108 Neutralization Solution...........................................................18 Neutrino................................................................................ 147 Nitro Blue Tetrazolium.......................................................75,88 Nitrocellulose.......................................................................... 89 Northern blotting.................................................................... 80 protocol............................................................................................ 81

Nuclean................................................................................. 140 Nucleic Acids.......................................................................... 33 Nucleotide............................................................................... 33 Nuclistain............................................................................27,79 Nuc-Wipes.............................................................................141 O

Liquid scintillation countingsamples

Ohm’s law............................................................................... 35 OptiClear...............................................................................161 OptiClear solvents................................................................ 160 Osmium tetroxide................................................................... 117 Oxidizing fixatives................................................................108

Liquiscint.................................................................................133 Low melting point agarose..................................................... 60

Oxosol 306...........................................................................137 Oxosol C14............................................................................137

Luminol..................................................................................... 88

Paraffin wax.....................................................................110,111 PBS (10X) Phosphate Buffered Saline....................................17 PCR Analysis........................................................................... 55 Peptide mapping..................................................................... 70

discrete samples............................................................................154

DNA purification.............................................................................62 in gel ligation...................................................................................63 in gel restriction digestion...............................................................63

MALDI MS Analysis from Insite stained gels.........................71 Mass spectroscopy electrophoresis.................................................................................46 protocol............................................................................................45

2-Mercaptoethanol.................................................................31 Mercaptoethanol.................................................................... 40

protocol............................................................................................70

Peroxidase................................................................................75 Peroxidase-antiperoxidase complex (PAP)......................... 116 Phosphors.............................................................................. 149 secondary......................................................................................149 structures and emission wavelengths (table)...............................149

Index of Subjects

sample preparation using the Insite System.................................. 71

Maxam & Gilbert Sequencing.............................................. 45

osmium tetroxide............................................................................117

Picric Acid Fixatives..............................................................108 pKa ..................................................................................... 38 USA: 1-800-526-3867 EUROPE: 441 482 646022

173 371

Alphabetical Index P

continued

Polyacrylamide....................................................................... 36 Polyacrylamide gels counting samples in.......................................................................156

Polymerase chain reaction..................................................... 55

gel electrophoresis of products......................................................56 in - gel..............................................................................................63 primer dimer...............................................................................55,56 protocol............................................................................................55

Polyvinylidine difluoride (PVDF)............................................ 89 Pore size.................................................................................. 36 agarose......................................................................................36,37 polyacrylamide...............................................................................36

Power (wattage)................................................................35,44 Primer extension...................................................................... 50 protocol............................................................................................50

Protein detection................................................................19,84 staining....................................................................................... 19,84 coomassie blue protocols............................................................85 Insite System.................................................................................85 membrane blot staining...............................................................87 powdered stains........................................................................... 21 products for post-electrophoretic visualization.......................... 19 silver stain.....................................................................................20 silver stain protocol......................................................................87 Sterling Rapid Silver Stain...........................................................20 Western blotting........................................................................ 87,89

Protein electrophoresis

buffers for protein electrophoresis.................................................... 6 denaturing protein electrophoresis: SDS-PAGE............................67 gel formulations............................................................................68 gel preparation............................................................................68 gradient gels.................................................................................70 gradient gels - protocol...............................................................69 sample preparation......................................................................67 tris-tricine gels...............................................................................68 troubleshooting.............................................................................94 gels for protein analysis.................................................................... 6 native protein electrophoresis......................................................... 74 Ferguson plots.............................................................................. 74 gel preparation............................................................................ 75 gradient gels................................................................................. 74 sample preparation...................................................................... 74 pre-mixed buffers............................................................................ 16

Protein Loading Buffer Blue (2X).............................................16 Protein purification...................................................................71

Index of Subjects

crush and soak................................................................................. 71 electroelution................................................................................... 71 using the Insite System..................................................................... 71

Proteins.................................................................................... 34 Protein Standards.................................................................... 25 ProtoBlock System................................................................... 24 ProtoGel................................................................................. 6,7 ProtoGel Buffers................................................................... 6,16 ProtoMarkers........................................................................... 25 ProtoMetrics............................................................................ 25 Pulsed field gel electrophoresis (PFGE)................................. 64 AquaPor ES...................................................................................... 15 Q

Quenching...................................................................... 150,151 channels ratio................................................................................ 151

174

USA: 1-800-526-3867 EUROPE: 441 482 646022

Q continued chemical.........................................................................................150 color ......................................................................................150,154 correction....................................................................................... 151 external standard.......................................................................... 151 R

Radiation safety.............................................................140,157 wipe testing............................................................................ 141,157

Radioactive decay................................................................ 147 detection and quantitation............................................................147 Becquerel....................................................................................147 counts per minute.......................................................................147 Curie...........................................................................................147 types of emissions..........................................................................147 alpha particles...........................................................................147 beta particles..............................................................................147 gamma rays................................................................................147

Radioactive emissions

Gamma rays Secondary áş&#x17E; emissions.............................................................147

Random Amplification of Polymorphic DNA (RAPD).......... 48 Reducing agents..................................................................... 40 Reptation.................................................................................. 64 Resolution polyacrylamide...............................................................................37

Restriction mapping.................................................................61 in - gel digestion..............................................................................63

Riboflavin..................................................................................31 Ribonuclease protection......................................................... 50 protocol............................................................................................50

Ribonucleosides...................................................................... 65 RNA electrophoresis............................................................... 65 gel preparation formaldehyde gels.......................................................................65 glyoxal gels..................................................................................65 sample preparation.........................................................................65

RNA mapping......................................................................... 49 S

S-1 mapping........................................................................... 49 protocol............................................................................................49

Salt wave................................................................................. 39 Sample preparation denaturing protein gels protocol.........................................................................................67 native DNA gels..............................................................................54 native protein gels........................................................................... 74 RNA ................................................................................................65

Sanger dideoxy sequencing.................................................. 46 electrophoresis.................................................................................46 protocol............................................................................................46 substrate preparation......................................................................46

Saponin.................................................................................. 115 Scintillation detection............................................................ 148 Scintillation Vials................................................................... 142 Scintillators............................................................................ 145 SDS ............................................................... 31,34,39,40,67 SDS-PAGE.......................................................................... 39,74

buffers for SDS-PAGE..................................................................... 16 gel matrices for SDS-PAGE...........................................................6,7

Secondary áş&#x17E; emissions........................................................ 147

continued

Sectioning...............................................................................110

electron microscopy............................................................... 110,117 positive staining...........................................................................117 frozen sections............................................................................... 110 paraffin blocks............................................................................... 110

SequaGel MD..........................................................................12 SequaGel XR............................................................................12 Silver staining protein protocol...............................................................................87 Sterling Rapid Silver Stain..............................................................20

Solid scintillation................................................................... 148 Solusol ................................................................................... 138 Solvents for liquid scintillation counting.............................. 148 Southern blotting..................................................................... 80 protocol............................................................................................82

SSCP analysis.......................................................................... 59 SequaGel MD................................................................................. 12

Stacking gel........................................................................39,67 Staining (histology).................................................................111 electron microscopy.......................................................................117 Hematoxylin and Eosin stain.........................................................113 histological stains...................................................................... 104 protocol........................................................................................113

Static electricity in LSC......................................................... 152 Sterling Rapid Silver Stain...................................................... 20 Submarine gels....................................................................... 60 Superoxide detection........................................................... 120 Diogenes........................................................................................120

Superoxide dismutase.............................................................75 Surfactants........................................................................ 40,151 T

TBE buffer

glycerol artifacts..............................................................................54

TBE Buffer (10X or 5X)............................................................18 TEMED................................................................................ 31,36 Tissue processing

continued

Troubleshooting

electrophoresis agarose gels.................................................................................93 denaturing DNA-PAGE gels.......................................................92 denaturing protein gels................................................................94 liquid scintillation counting...........................................................158

TTE buffer................................................................................. 47 TTE Glyerol Tolerant Buffer (20X)...........................................18 Tube gels...................................................................................41 Tween-20.................................................................................31 Two dimensional electrophoresis........................................... 77 U

Uniscint BD.....................................................................130,135 Uracil interference assay...................................................51,53 protocol............................................................................................53

Uranyl acetate........................................................................ 117 Urea .................................................................... 31,40,43,44 UreaGel 6 and 8.................................................................... 10 V

Vacuum blotting...................................................................... 80 Vertical gels..............................................................................41 W

Waste issues.......................................................................... 156 Wedge gel.............................................................................. 46 Western blotting.................................................................22,89 blocking............................................................................................89 pre-mixed buffers...................................................................... 16,24 products............................................................................................22 protocol............................................................................................90

Wipe testing.......................................................................... 157 Nuc-Wipes.................................................................................... 141 X

Xylene ............................................................................109,151 Xylene Cyanole FF.................................................................. 28

clearing......................................................................................... 109 Histo-Clear................................................................................. 110 xylene........................................................................................ 109 dehydration................................................................................... 109 electron microscopy.......................................................................117 schedule........................................................................................ 109

Tissue samples (LSC)............................................................ 155 TLC plates.............................................................................. 155 counting scraped samples............................................................155 fluorography..................................................................................155

Index of Subjects

Toluene.....................................................................111,148,151 Tracers ................................................................................... 147 Tracking dyes.......................................................................... 54 Trailing ions............................................................................. 39 Transmission electron microscopy......................................... 116 Tricine ......................................................................................31 Triple Dye Loading Buffer (6X)...............................................18 Tris.............................................................................................31 Tris-Glycine Electroblotting Buffer (10X)................................17 Tris-Glycine-SDS PAGE Buffer (10X).....................................16 Tris-Tricine-SDS PAGE Buffer (10X)........................................16 Triton-X100.............................................................................151 USA: 1-800-526-3867 EUROPE: 441 482 646022

175 571

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