2012/13
Research Product Catalogue ww.buybbi.com
1. About BBI
2
2. Introduction to Gold Labelling
4
3. Colloids
7
4. Gold Colloid Products and Pricing
12
5. Silver Colloid Products and Pricing
13
6. Microarray Gold and Silver Products and Pricing
14
8. High OD Gold Colloid
15
7. How to Choose a Gold Conjugate
16
9. EM Grade Gold Conjugates
19
10. Gold Conjugate Products and Pricing
21
11. GoldLink – Rapid Conjugation Kit
23
12. Getting the Best from Immunogold Labelling
25
13. Gold Stains Products and Pricing
27
14. Silver Enhancing Products and Pricing
31
15. Unicryl Products and Pricing
33
16. Ancillary Products and Pricing
34
17. How to Order
36
18. BBI Distributors
37
About BBI Distribution
Ordering
Labelling
Gold Conjugates
High OD
Microarray
Silver Colloid
Gold Colloid
Contents
1
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About BBI
About BBI
Gold Colloid
Technical support
Silver Colloid
We offer a high level of technical support. Our inhouse team is always available to offer help and troubleshooting advice, on the use of nanoparticles and conjugates. To give greater assistance in choosing and using our products, we have included a section on the choice of gold conjugate depending on application and requirements. We also have a range of additional technical information, including technical booklets and protocols. This information is available on request. Additionally our Technical Support department can be contacted on +44 (0) 29 2074 7232 or e-mail at info@buybbi.com
Microarray
Collaboration
High OD
BBInternational is a leading manufacturer of high quality gold and silver nanoparticles and reagents for a range of research, diagnostic and nanotechnology applications. Our products, which are manufactured to a proprietary technique, are subject to a number of stringent quality control measures.
We collaborate with many laboratories around the world. In our laboratories we have a number of ongoing programmes using nanoparticles and reagents in various applications for the development and application of new products. Many of the images and data that appear in our literature have arisen from these joint collaborations. We welcome any requests for collaboration on new techniques, products or applications.
Gold Conjugates
High Quality Products
Labelling
Our products are manufactured and tested with the highest possible emphasis on reliability and reproducibility. Every product is rigorously tested with the most stringent quality assurance procedures. Because of this policy we now offer the widest range of top quality colloidal gold and silver nanoparticles, reagents and associated products world-wide.
Ordering Distribution
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2
About BBI
Products Overview
Gold Colloid
Gold Colloid
Silver Colloid
BBI’s unique recipe gold colloid (colloidal gold) has been used for over 25 years due to its superior quality and performance. The optical properties of gold at the nanoscale means there is a variation in colour from red to pink to orange depending on nanoparticle size, a property that can be successfully exploited in a range of applications. BBI’s colloidal gold offers excellent stability and sensitivity characteristics, and as the market has grown to expect higher levels of sensitivity, gold has increasingly become regarded as an extremely reliable raw material in providing an accurate visual signal.
Microarray
Silver Colloid
High OD
The use of silver can potentially lead to improved sensitivity where silver nanoparticles demonstrate a stronger, more distinctive SPR spectrum than the corresponding gold particles. Use of silver in place of gold can potentially result in a more defined wavelength shift, and broadens the range of optical properties available – an advantage for applications where multiplexing is needed.
Gold Conjugates
Gold Conjugates
A colloidal gold conjugate consists of a suspension of gold particles coated with a selected protein or macromolecule (such as an antibody). BBI has a range of EM grade products, and LM grade products, that combine different antibodies on the range of particles predominantly, from 2, 5, 10, 15, 20, 30 & 40nm.
High OD Gold Colloid can be custom made to your specific requirements. Whether you require 40nm particles at 40OD, 150nm particles at 150OD or 250nm particles at 250OD, our highly experienced technical team are able to help.
Products and Services for Diagnostics In addition to the research products listed in this catalogue we also offer end to end OEM diagnostic services including: • Bulk supply of gold and silver colloid and conjugates • Custom conjugation of proteins and oligonucleotides • Research and development of rapid diagnostic tests • Rapid molecular diagnostic platforms • Contract manufacture • Assay manufacturing machinery
Custom Conjugation Service
Ordering
Labelling
Our many years of expertise in manufacturing and using gold and silver nanoparticle reagents has enabled us to provide a comprehensive service for custom conjugation of products not listed in the Catalogue. We have the capability to work with a range of proteins, including antibodies and antigens as well as the more complex area of nucleic acid conjugation. We also offer conjugation to other types of particle including; latex, carbon and polymers.
High OD Gold Colloid
Distribution
BBI’s high OD gold can be used to provide dense uniform mono-layers of gold particles on substrates and can also simplify the conjugation procedure by removing the concentration steps. This saves time, reduces costs and ensures waste is minimised.
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About BBI
Introduction to Gold Labelling
Conjugation The conjugation of selected proteins to gold particles depends upon at least three physical phenomena:
a) Charge attraction of the negative gold particle to positively charged protein
Microarray
b) Hydrophobic adsorption of the protein to the gold particle surface c) Binding of the gold to sulphur (dative binding) where this may exist within the structure of the macromolecule.
High OD
Gold Colloid Pro oteins Gold Conjugates
A very important use for gold conjugates has emerged in their incorporation into rapid test immunoassays. In these techniques the unique red colour of the accumulated gold label, when observed by lateral or transverse flow along a membrane on which an antigen is captured, or by observation of the red colour intensity in solution, provides an extremely sensitive method for detecting sub nanogram quantities of proteins in solution.
For detection by eye, gold particles will also reveal immobilised protein on a solid phase such as a blotting membrane through the accumulated red colour of the gold sol. Silver enhancement of this gold precipitate also gives further sensitivity of detection.
Silver Colloid
Over 50 years ago fluorescent labels were conjugated with antibodies for the first microscopical detection of antigens in tissues. In 1970 enzyme labels first provided a permanent coloured stain with higher resolution and greater stability than fluorescent signals. With the advent of gold labels in 1971, sensitive high resolution immunocytochemistry became possible in the electron microscope for the first time. The subsequent development of silver enhancing methods then allowed gold labels to provide a highly specific and sensitive method for visualising antigens in light microscopy. Since their introduction to microscopy, gold labels have also become recognised as very important tools for detection and quantitation of proteins, antigens, and nucleic acids when used with other techniques such as blotting, flow cytometry, hybridisation, and fingerprint identification.
Gold Colloid
History
Adsorption of proteins to gold colloid. Gold may be conjugated to a wide variety of molecules including proteins (e.g. antibodies), polypeptides, carbohydrates, polymers, polysaccharides, enzymes and nucleic acids. The conditions under which a gold conjugate is manufactured drastically affect its performance and stability.
Distribution
Under an electron microscope the particles are visible without further treatment. Under a light microscope, however, the gold particles are made visible through a short and simple silver enhancing procedure.
Stabilised Gold Colloid Ordering
Gold labels act as markers for molecules that are otherwise invisible by eye or through other detection systems. A colloidal gold conjugate consists of a suspension of gold particles coated with a selected protein or macromolecule (such as an antibody). The gold particles may be manufactured to any chosen size from 2-500nm. This gold probe detection system, when incubated with a specific target, such as in a tissue section, will reveal the target through the visibility of the gold particles themselves.
Labelling
Gold Probes
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About BBI High OD
Microarray
Silver Colloid
Gold Colloid
Introduction to Gold Labelling Why Gold?
What Determines High Quality?
There are several advantages in choosing gold particles as markers for microscopic and non-microscopic identification of target molecules.
Several essential features must apply to gold probes if they are to be considered reliable high quality reagents for experimental and diagnostic research.
The choice of particle size makes it possible to study antigens microscopically over a wide range of magnifications and provides the opportunity for multiple labelling of antigens on tissue sections. In the electron microscope labelling with gold particles is completely unequivocal. The label cannot easily be mistaken for any other tissue structure and is either present or not present. The resolution of this labelling in both Electron Microscopy and Light Microscopy is thus higher than with any other method.
Antibodies and Proteins
While fluorescent labels and enzyme based colour reactions may fade with time and light exposure, gold particles do not disappear but give a permanent label.
BBInternational has perfected a proprietary technique for the manufacture of mono-disperse gold and silver colloids, to pre determined sizes with a coefficient of variation (CV) of less than 8% of particle size. This ensures our gold conjugates are consistent from batch to batch and give the same high performance time after time. In addition each batch is stringently quality controlled to ensure our high specifications are adhered to.
Gold Conjugates
Because of the particulate nature of the gold label, quantitation in the electron microscope is possible, even for more than one antigen by multiple labelling with different particle sizes.
Labelling
In the light microscope, the intense brown/black stain produced by silver enhancement of the gold signal has been shown to give a greater overall sensitivity and far greater resolution than other immunocytochemical methods and tissues can be counterstained with all the usual staining procedures.
Ordering
Unlike some enzyme based detection systems; gold probes are essentially non toxic, safe and easy to use. The good stability of a well made gold conjugate gives the product a long shelf life whether frozen or stored at 2-8°C.
Distribution
For the staining of proteins immobilised onto membranes, gold probes have unsurpassed detection sensitivity and resolution.
To begin with the raw materials, all antibodies and proteins used for conjugation are recommended to be affinity purified and of the highest quality. They must possess very strong affinity for the specific antigen and have high avidity to withstand severe incubation and washing conditions. Cross reactivity must be the lowest possible. In order to ensure consistent and reliable results, BBInternational uses only the highest quality antibodies and proteins for reagent manufacture.
Sensitivity and Stability Sensitivity is all important in immunolabelling for detection of low levels of antigens in cells and tissues. In order for a gold probe to produce a strong specific signal together with a low background the conjugation of antibody to gold particle must be complete and stable under a variety of incubating conditions. The gold particle must have the minimum effect on the activity of the antibody to which it is conjugated but be strongly enough absorbed to the surface to remain stable for years. For long term storage conjugates are made using glycerol based making it possible to freeze a gold conjugate without loss of the antibody activity. All standard BBInternational gold conjugates may be frozen and stored over long time intervals without deterioration.
Finally, and perhaps surprisingly, gold probes are inexpensive in their application and their high sensitivity often allows valuable primary antibodies to be diluted significantly further than with other systems.
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About BBI
Introduction to Gold Labelling
Gold Colloid
Clustering
Silver Colloid
For electron microscopy a very low level of clustering is important, especially if quantitation is to be performed or high quality micrographs are required. Clustering indicates a poor absorption of protein with bridging between gold particles. Heavily clustered conjugates have low stability and poor sensitivity. At the very least gold conjugates should have greater than 85% singlets. All BBInternational EM Grade gold conjugates are made to this high specification.
Microarray
Quality Assurance
Gold Conjugates
Antibody Purification
Determine Antibody Titre
Labelling
CREATIVE CONJUGATION SERVICE BBI are experts in creative conjugation.
High OD
Rigorous testing and fully documented QC protocols ensure that all of our products meet tight specifications, thus ensuring the highest possible consistency and performance. It is this focus on quality control, coupled with our unique manufacturing process that has resulted in BBInternational colloids being recognised as the ‘Gold Standard’ within the Life Science and Lateral Flow diagnostics industry.
Let us conjugate your antibody to gold colloid.
Talk to us about your requirements
Column Chromatography
Distribution
Tel: +44 (0) 29 2074 7232 info@buybbi.com
Manufacture and Concentrate Conjugate Ordering
• 3ml finished conjugate • Technical advice and ongoing support
Binding Analysis
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About BBI Gold Colloid
Colloids Gold Colloids
Silver Colloid
The full range of BBI high quality gold conjugates is listed in this Catalogue. However, we recognise that there may be occasions when customers may need to use gold conjugates other than those listed here. Solutions of unconjugated colloidal gold sols are available from BBI in a wide range of particle sizes. These colloids may be conjugated to antibodies, proteins, or many types of macromolecule for binding and reaction labelling studies.
High OD
Microarray
The same high quality manufacturing principles are used for the production of these gold sols as those used in the preparation of BBI gold conjugates listed in this Catalogue. Colloids are made with a very narrow size distribution, the actual size being measured by electron microscopy and provided on the data sheet with each product. The colloids are provided in sterile containers ready for conjugation. They will remain stable until the use by date if stored unopened at 4°C. Do not freeze.
b) Electron Microscopy For electron microscopy any particle size may be used, the smaller particles giving the highest labelling efficiency. The magnification required for the EM study determines the particle size to be used. Smaller particles may be silver enhanced for viewing at lower magnifications. For scanning electron microscopy 20nm, 30nm or 40nm may be used together with backscattered electron imaging. However, smaller particles will give a higher labelling intensity and may be silver enhanced. c) Blotting For blotting applications the larger particles give the most visible stain without silver enhancing. Larger particles may produce more steric hindrance to the labelling, however. The choice lies between 20-40nm for many applications. For use with the BBI Blotting Silver Enhancing Kit any particle size may be used, but again smaller particles will give the highest labelling intensity. For silver enhancing we recommend 2nm or 5nm gold particles.
Gold Conjugates
Unit Size
Labelling
BBI gold colloids are available in 20ml, 100ml and 500ml volumes in polycarbonate bottles. Please indicate the required volume when ordering. Please refer to the current price list from your local distributor.
Ordering
Which Gold Colloid to Choose?
Distribution
The complete range of particle sizes of BBI gold colloids is given below. The choice of particle size depends on the specific application. Please also refer to the preceding Sections for applications in EM, LM or Blotting applications.
BBI gold colloids of 20nm and above are supplied at optical density 1.0 measured at 520nm. 5, 10, and 15nm are supplied at approximately OD0.8. 2nm colloids are approximately OD0.02 at 400nm. Colloids contain 0.01% concentration of HAuCl, except EM.GC2 which has a concentration of 0.001%.
Bulk Purchase of Gold Colloids Larger volumes of gold colloids of any chosen particle size can be supplied for bulk purchase. Please contact us for details by emailing info@bbigold.com.
a) Light Microscopy For light microscopy, in conjunction with silver enhancing, the smaller sizes are recommended (2nm and 5nm).
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About BBI
Gold Colloid Supporting Data
Gold Colloid
Zeta Potential (mV)
2
1.50E+14
1.21-05
-34.1
5
5.00E+13
6.32-05
-38.6
10
5.70E+12
5.76-05
-38.4
15
1.40E+12
4.77-05
-42.8
20
7.00E+11
5.66-05
-41.4
30
2.00E+11
5.46-05
Data currently not available
40
9.00E+10
5.82-05
-44.0
50
4.50E+10
5.68-05
Data currently not available
60
2.60E+10
5.68-05
-50.2
80
1.10E+10
5.69-05
-52.8
100
5.60E+09
5.66-05
-56.3
150
1.66E+09
5.66-05
-60.5
200
7.00E+08
5.66-05
-56.1
250
3.60E+08
5.66-05
-66.7
High OD
Mass of gold per ml (g)
Microarray
No. particles per ml
Silver Colloid
Particle Diameter (nm)
Wavelength a scans (600-450nm) of Gold Colloid Life Science Starter Kit 1.2
5nm = 517.0 10nm = 518.2 20nm = 52 20 524 4.8 8 40nm = 526.4
20nm
10nm
40nm
0.6
0.2
0
430
Labelling
04 0.4
Wavelength a scans (600-450nm) of Gold Colloid Diagnostic Starter Kit 1.4
450
470
490
510
530
550
570
590
Peak Wavength (nm)
1.2
610
1
Ordering
20nm = 524.8 40nm = 526.4 60nm = 535.4 80nm = 550.0
Wavelength (nm)
20nm
Absorbance
Absorbance s
0.8
5nm
Gold Conjugates
1
Peak Wavelength ( ) (nm)
0.8
40nm 60nm 80nm
0.6
Distribution
0.4 0.2 0 430
450
470
490
510 530 550 Wavelength (nm)
570
590
610
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8
About BBI Gold Colloid
Gold Colloid Applications The table below identifies various areas of application for gold nanoparticles and the functions that these particles play within this area of research. These range from the original design uses such as immunohistochemistry and lateral flow diagnostics, to drug delivery, chemical sensing and even forensic science and textile printing and development.
Nanoparticle Size Range
Areas of Use
Function
Small (2nm-15nm)
Immunohistochemistry, Light Microscopy, High Magnification TEM, Drug Delivery, Biomarkers, Gas Sensors, Coatings
• Silver enhanced antibody detections within cells • Solvent suspensions in gas detection • Decorative coatings in paint and textile industries • Thin film substrate coatings for semi-conductor manufacture or electrochemical devices • Tumour targeting in cancer for drug delivery or identification of substrates
Medium (20nm-60nm)
Lateral Flow Assays,TEM, SEM, Environmental Detection and Purification, Data Storage, Drug Delivery, Biomarkers, SERS, Photothermolysis, Catalysis Chemical Sensors, DNA Detection
• Mercury detection and removal from contaminated water • Fluorescent probes • Molecular imaging agents • Colorimetric competition assays • Glucose biosensors • Single nucleotide polymorphism detection • Tumour cell targeting and imaging • Chemical reduction catalysis • Creation of C-C bonds and oxidation of organic substances
Large (80nm-250nm)
Flow Cytometry, Printed Text, Conductive Films, SERS, Forensic Science, Electronic Device Manufacture, SERS, Optical Mammography
• Detection of CD4 cells using flow cytometry • Fuel cell efficiency • Support of silk fibroin fibres for use in the textile industry • Direct printing of gold particles onto paper and fabric • Detection of biomarkers in cells for imaging and sensing purposes
Distribution
Ordering
Labelling
Gold Conjugates
High OD
Microarray
Silver Colloid
Today’s world is connected at the touch of a button and we live in a society that thrives on knowledge. If that knowledge can be passed to health experts, they then have the opportunity to complete fast, frequent, reliable and cost effective diagnosis to help deliver effective and efficient treatment to patients.
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About BBI Microarray High OD
These differing colours allow the labelling of different analytes with these various noble metal nanoparticles. BBI has the capacity and experience to produce high volumes of silver colloid at exceptionally high quality standards, allowing multiplexing applications within lateral flow tests.
BBI’s silver colloid is available in 20, 40, 60 and 80nm particle sizes. The colloidal silver is made according to BBI’s unique recipe and offers superb quality and performance. The colloid is produced by a wet chemical method and uses citrate as a stabilising agent. The particles possess a citrate shell which gives them a net negative charge. This allows the nanoparticles to repel each other and stay in solution. For larger particle sizes (60 and 80nm) the surface charge is unable to prevent the particles aggregating together and settling at the bottom of their container. However, the particles can be re-suspended into solution simply by inverting the bottle and gentle shaking. BBI has developed a unique recipe silver colloid; allowing for precise control of the particle size, monodispersity and large batch sizes of up to 56L, at standard concentration.
Silver Colloid
20nm silver colloid shows a pronounced absorption peak around the 400nm wavelength of light. This is in contrast to gold colloid which exhibits an absorption peak around the 520nm wavelength. This results in silver colloid possessing an intense yellow colour as opposed to the characteristic ruby red colour of gold colloid and the black colour produced by platinum colloid.
Gold Colloid
Optical Properties Characteristics of Colloidal of BBI Silver Silver Colloid
0.9 80nm
60nm
0.8
40nm
Absorbance
0.6 0.5 0.4
Labelling
0.3
Gold Conjugates
20nm
0.7
In summary, the interesting features of colloidal silver such as its antibacterial effects and unique optical properties, along with recent advances in fields such as SERS and electronics manufacture, make it a colloid with a multitude of uses. In order to get the best results from a silver colloid, researchers and engineers need a high quality, consistent and monodisperse particle. BBI’s silver colloid offers superb performance with a large batch size and is suitable for use in a wide range of applications.
0.2 0.1 0 320 350
375
400
425
525
550
575
600
Ordering
450 475 500 Wavelength (nm)
Distribution
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About BBI Gold Colloid
Colloids Additional Notes
High OD
Microarray
Silver Colloid
The colloids manufactured by BBI are for the purpose of conjugation to macromolecules, (antibodies, antigens, proteins, etc.), and BBI does not guarantee the suitability for any other purpose. Optimising the conjugation of proteins is a complex, and for the inexperienced, a time consuming process. BBI have many years of experience in this respect, and offer a custom conjugation service, which will give you an optimised conjugation within a short time frame. Although the cost of this service is higher than a bottle of gold, the development time saved will offset this cost. To discuss your requirements, please contact our custom conjugation department, (email: info@bbigold. com). It should also be noted that not all proteins are suitable for conjugation to gold colloid. If you are unsure about the suitability of these colloids for your purpose, please contact us at the above email address and we will be pleased to discuss your application.
Gold Conjugates
If a bottle of colloid is left undisturbed for a period of time, the colloid will start to settle out and a ring of gold particles will appear in the bottom of the bottle. Again this is normal and can be easily resuspended by inversion of the bottle several times.
Bottles should only be opened in a clean room, preferably in a laminar flow cabinet. Remove the colloid by pouring out what you need. Never place a pipette or anything in the bottle that can introduce contamination. pH of colloids can be adjusted by using 1% or 0.1% HCl or NaOH solutions. Note that at extremes of pH, the colloid will collapse. Never add high molarity buffer salts to the colloid, without checking a small volume first. Colloid will turn purple / blue on contact with high molarity salts, and precipitate out of solution. Colloid damaged in this way will not be replaced as part of the guarantee. Colloids will keep well for many months unopened and are guaranteed until the expiry date unopened. Once opened, they may start to deteriorate and eventually precipitate. For this reason purchase only what you will need to use immediately, and discard any remaining colloid one month after opening. Purchasers of large volumes of colloid may like to receive their colloids packaged in convenient sized containers. Please contact our technical department to discuss your requirements.
Distribution
Ordering
Labelling
Colloids should be stored correctly to prevent them from precipitating. They should be stored between 2-8°C, although for short term storage the is no problem with room temperature. On no account should the colloid be allowed to freeze. Even partial freezing of the colloid will cause it to precipitate out of solution. This results in a clear solution with black lumps of precipitate in the bottom of the bottle. Therefore when storing the colloid do not place it close to the plate at the back of a fridge, or directly in front of the cold air in a cold room. Colloids damaged in this way will not be replaced as part of the guarantee.
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About BBI
Gold Colloids
Pricing Product Gold Colloid
20ml
100ml
500ml
£37.00
£146.00
£487.00
Array Gold Colloid
See p.14 Silver Colloid
Sizes Size
Code
Size
EMGC2 EMGC5 EMGC10 EMGC15 EMGC20 EMGC30 EMGC40
2nm 5nm 10nm 15nm 20nm 30nm 40nm
EM EMGC50 MGC C50 0 EMGC60 EM MGC C60 0 EMGC80 EM MGC C80 0 EMGC100 EM MGC C10 00 EMGC150 EM MGC C15 50 EMGC200 EM MGC C20 00 EMGC250 EM MGC C25 50
50nm 60nm 80nm 100nm 150nm 200nm 250nm
High OD
Code
Microarray
• Lowest available coefficient of variation (CV) • The choice of particle size makes it possible to study antigens microscopically • Allows multiple labelling of antigens on tissue sections • Labelling is completely unequivocal • Provides a permanent label • Quantitation under the electron microscope is possible due to its particulate nature • The optical properties mean there is a variation in colour from red to pink to orange depending on particle size, a property that can be successfully exploited in a range of applications • Known to be catalytically active for a range of commercially important reactions
Gold Colloid
BBI’s unique recipe gold colloid has been market leading for over 25 years due to its superior quality, consistency and performance, offering excellent stability and sensitivity characteristics. There are several advantages of choosing BBI’s gold colloid:
Gold Conjugates
Gold Colloid Starter Packs
Labelling
A wide range of particle sizes can be conjugated to proteins and macromolecules, and first time gold users or those trying out a new process may be unsure of which size to purchase. Our gold colloid starter packs provide a range of particle sizes allowing a comprehensive evaluation.
Volume
Price
Gold Colloid 5nm/10nm/20nm/40nm
GCIKITLIFE
20 ml o off ea each ach
£13 £131.00 31.0 00
Gold Colloid 20nm/40nm/60nm/80nm
GCIKITDIAG
off ea each 20 ml o ach
£131.00 £13 31.0 00
Gold Colloid 20nm/40nm/60nm/80nm
GCKITDIAGPLUS
each 100 0 mll of e ach
£440.00 £44 40.0 00
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Distribution
Code
Ordering
Product
12
About BBI Gold Colloid
Silver Colloid
Silver Colloid
The use of silver can potentially lead to improved sensitivity where silver nanoparticles demonstrate a stronger, more distinctive SPR spectrum than the corresponding gold particles. Use of silver in place of gold can potentially result in a more defined wavelength shift, and broadens the range of optical properties available – an advantage for applications where multiplexing is needed.
Pricing Product
20ml
100ml
500ml
Silver Colloid
£37.00
£146.00 £1 146.0 00
£487.00 £4 487 7.0 00
Array Silver Colloid
See p.14
Code
Size
Code
Size
EMSC20 EMSC40
20nm 40nm
EM EMSC60 MSC C60 0 EM MSC C80 0 EMSC80
60nm 60 0nm m 80 0nm m 80nm
Distribution
Ordering
Labelling
Gold Conjugates
High OD
Microarray
Sizes
13
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About BBI
Microarray Gold & Silver Colloid
Gold Colloid
In the Microarray market, gold with its unique properties has emerged as an alternative to fluorescence detection.
Key Features • Highly Specific • Reproducible • Stable
Silver Colloid
BBInternational’s Microarray gold is the ideal solution for overcoming many of the problems associated with fluorescence detection methods, including low sensitivity, signal intensity, reduction due to photochemical bleaching, quenching and expensive image analysis systems.
• Highly Sensitive • Reliable • Perfectly Spherical Shapes (≤95 %) • Low CV (<8%)
Size
Volume
ArrayGC2
2nm
20ml 20ml
ArrayGC5
5nm
2 20ml 0ml
ArrayGC10
10nm
20ml 20ml
ArrayGC15
15nm
20ml 20ml
ArrayGC20
20nm
20ml 20ml
ArrayGC30
30nm
20ml 20ml
ArrayGC40
40nm
2 20ml 0ml
ArrayGC50
50nm
20ml 20ml
ArrayGC60
60nm
20ml 20ml
ArrayGC80
80nm
2 20ml 0ml
ArrayGC100
100nm
2 20ml 0ml
ArrayGC150
150nm
20ml 20ml
ArrayGC200
200nm
20ml 20ml
ArrayGC250
250nm
20ml 20ml
Code
Size
Volume
ArraySC20
20nm
2 20ml 0ml
ArraySC40
40nm
2 20ml 0ml
ArraySC60
60nm
2 20ml 0ml
ArraySC80
80nm
2 20ml 0ml
Microarray
Code
Pricing Product
High OD
Microarray Gold Colloid Microarray Silver Colloid
20ml £110.00 £110.00
Gold Conjugates Labelling Ordering Distribution
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14
About BBI Gold Colloid
High OD Gold Colloid
Silver Colloid
High OD gold can be used to provide dense uniform mono-layers of gold particles on substrates and can also simplify the conjugation procedure by removing the concentration steps. This saves time, reduces costs and ensures waste is minimised.
Particle Diameter
No. Particles per ml
No. Moles Particle per ml
Molar Particle Concentration (No. moles per L)
15
Mass of Gold per ml (g)
Moles of Gold per litre
1
7.00E+11
1.1624E-12
1.1624E-09
5.66E-05
2.87E-04
20
5
3.50E+12
5.81202E-12
5.81202E-09
2.83E-04
1.44E-03
20
10
7.00E+12
1.1624E-11
1.1624E-08
5.66E-04
2.87E-03
20
15
1.05E+13
1.74361E-11
1.74361E-07
8.49E-04
4.31E-03
20
50
3.50E+13
5.81202E-11
5.81202E-07
2.83E-03
1.44E-02
20
100
7.00E+13
1.1624E-10
5.81202E-05
5.66E-03
2.87E-02
40
1
9.00E+10
1.49452E-13
1.49452E-10
5.82E-05
2.96E-04
40
5
4.50E+11
7.4726E-13
7.4726E-10
2.91E-04
1.48E-03
40
10
9.00E+11
1.49452E-12
1.49452E-09
5.82E-04
2.96E-03
40
15
1.35E+12
2.24178E-12
2.24178E-09
8.73E-04
4.43E-03
40
50
4.50E+12
7.4726E-12
7.4726E-09
2.91E-03
1.48E-02
40
100
9.00E+12
1.49452E-11
1.49452E-08
5.82E-03
2.96E-02
60
1
2.60E+10
4.3175E-14
4.3175E-11
5.68E-05
2.88E-04
60
1
1.30E+11
2.15875E-13
2.15875E-10
2.84E-04
1.44E-03
60
1
2.60E+11
4.3175E-13
4.3175E-10
5.68E-04
2.88E-03
60
1
3.90E+11
6.47625E-13
6.47625E-10
8.51E-04
4.32E-03
60
1
1.30E+12
2.15875E-12
2.15875E-09
2.84E-03
1.44E-02
60
1
2.60E+12
4.3175E-12
4.3175E-09
5.68E-03
2.88E-02
80
1
1.10E+10
1.82664E-14
1.82664E-11
5.69E-05
2.89E-04
80
1
5.50E+10
9.13318E-14
9.13318E-11
2.85E-04
1.44E-03
80
1
1.10E+11
1.82664E-13
1.82664E-10
5.69E-04
2.89E-03
80
1
1.65E+11
2.73995E-13
2.73995E-10
8.54E-04
4.33E-03
80
1
5.50E+11
9.13318E-13
9.13318E-10
2.85E-03
1.44E-02
80
1
1.10E+12
1.82664E-12
1.82664E-09
5.69E-03
2.89E-02
Distribution
Ordering
Labelling
Gold Conjugates
High OD
Microarray
20
OD@520nm
BBIâ&#x20AC;&#x2122;s high OD Gold Colloid can be custom made to your specific requirements. Whether you require 40nm particles at 40OD, 150nm particles at 150OD or 250nm particles at 250OD, our highly experienced technical team are able to help. Please enquire for pricing.
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About BBI
How to choose a Gold Conjugate
Silver enhancing Primary antibody Secon ndary antibody
Direct
High OD
The indirect method is the most common for studying cells and tissues and avoids the need to label every primary antibody. It provides a universal method for detecting any primary antibody from the same primary species, i.e. all Rabbit antibodies may be detected by Goat anti-Rabbit gold conjugates, etc. The indirect method is longer than the direct method because of the need for two (or three) separate incubations but is the more widely used. The previous table indicates secondary gold labelled antibodies to be used with any chosen primary.
Microarray
Direct or indirect labelling (antibodies)
Silver Colloid
An extension of the indirect method may be used where there is no appropriate gold labelled secondary to match the primary antibody. For example if the unlabelled primary is from Pig and there is no matching gold labelled anti-Pig then an unlabelled anti-Pig (say Rabbit anti-Pig) may be used as a second step followed by a gold labelled third antibody (say Goat anti-Rabbit) that will detect the second antibody.
Gold Colloid
The aim of an antibody incubation with a specimen, whether it be a section, cells in suspension, a tissue slice, a blotted membrane, or a rapid diagnostic test assay, is to achieve the most intense specific signal and the least non specific background possible. The next section; ‘Getting the Best from Immunogold Labelling’ section describes this in detail but the initial choice of gold conjugate is also important.
Absorption with serum proteins Indirect
Gold Conjugates
Antibody gold conjugates are absorbed against certain species of serum proteins. This absorption reduces cross reactions to those species to an absolute minimum.
Silver enhancing of direct or indirect labelling.
When to use F(ab’) fragments?
When to use antibodies, Protein A, Protein G or Protein A/G?
Ordering
Protein A (PAG), Protein G (PGG) and Protein A/G (AG) are all separate proteins and will mimic a secondary antibody by binding to the Fc part of a primary antibody. In some situations they may be used in place of the secondary antibody. Unlike secondary antibodies, however, it is believed that there is only a single binding site for these proteins on the primary lgG, They do not bind with great affinity to IgM or lgA molecules.
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Distribution
The choice of the most appropriate system depends upon the type of component to be detected and what binding proteins are available. Some guidelines on making this choice are as follows:
In some applications background labelling may be a problem due to the attraction of the Fc region of the antibody gold conjugate to tissue components (called Fc receptors). Normally this is blocked by the simple application of normal serum prior to the first antibody. If the problem persists, however, then gold labelled F(ab’) fragments of antibodies may be used. These conjugates are listed in each Section.
Labelling
Gold conjugates may be used directly or indirectly to label antigens as indicated above. In the direct method the gold is conjugated to the primary antibody, whether monoclonal or polyclonal. This allows a single incubation to be performed and provides the simplest detection system. In the indirect method a primary unlabelled antibody is applied to the specimen to locate the antigen. This is followed by a gold labelled secondary that detects the primary antibody. This gold labelled secondary antibody is almost always an affinity purified polyclonal. In this way an amplification of the signal is achieved, often up to 10x compared with a direct incubation, according to the particle size.
16
About BBI Gold Colloid
How to choose a Gold Conjugate
Silver Colloid
The advantage of these proteins is that they provide a universal second step for a wide range of primary antibody species. They each bind with varying affinities to IgG molecules of different species as shown in the table. However, if possible we would always recommend the selection of a species specific secondary antibody in preference to Protein A, G or A/G.
Specific labelling
Microarray
Whole 1gG G
Non Specific N p labelling P Primary Antigen
High OD
Fc receptor
Gold Conjugates
F(ab ( 1)2 fragment
Second antibodies provide an amplified signal compared with Protein A.
Labelling
Secondary S antibody: a tib d g gold
Primary Antigen n
Ordering
For the indirect detection of a biotinylated primary antibody or nucleic acid probe there is a choice of Streptavidin or Goat anti-Biotin gold conjugates. Historically streptavidin has been the most frequently used detector for biotin because of the very high affinity constant between them. Most recently, however, Goat anti-Biotin has been shown to be a rather more sensitive detector of biotin compared to streptavidin when conjugated to gold particles. This is because of the relatively large molecular size of the antibiotin molecule (160,000 Daltons) compared to streptavidin (40,000 Daltons) and the distance between the binding of the gold on the Fc, from the binding region of the antibody F(ab’). This is especially so when using larger gold particles but becomes insignificant for 5nm and 1nm gold conjugates. On occasions where background from nonspecific attraction of goat antibodies may cause a problem streptavidin may provide a cleaner result. We recommend beginning with Goat anti-Biotin gold conjugates for the detection of biotin.
Cationic Gold
No attraction
Protein A: gold
Distribution
Primary
Streptavidin or Goat anti-Biotin?
Cationic gold allows highly sensitive and discrete microscopical studies of anionic (i.e. negative) sites in cells and tissues. The gold conjugate is made by careful conjugation to poly-L-lysine, a highly positive amino acid chain. A simple one step incubation of sections with the diluted Cationic Gold conjugate reveals subcellular sites having net negative charge. The charge distribution can be seen at a range of magnifications by using Cationic Gold of different particle sizes. Most cells of eukaryotic origin have a net negative surface charge from anionic plasma membrane components. This charge distribution is thought to be important in movement of various soluble macromolecules across cell walls. Thus the role of the surface charge in cellular behaviour through interaction with neighbouring cells can be studied. For in vitro and in vivo studies Cationic Gold may also be used effectively to study the uptake of anionic material by endocytosis.
Use of F(ab’)2 fragments to avoid non specific labelling.
17
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About BBI
How to choose a Gold Conjugate
High OD
d) Blotting For blotting applications where the gold conjugates are to be applied to proteins immobilised on nitrocellulose membranes, the choice is between 2nm and 20nm EM Grade gold conjugates. For ultra high sensitivity 2nm conjugates are preferred in combination with silver enhancing. However the 20nm gold conjugates allow blotted proteins to be identified on the immobilising membrane without silver enhancing. The 2nm and 5nm gold conjugates, because of their much reduced steric hindrance and greater particle density in solution, may be diluted much further to provide the same final intensity compared with the 20nm conjugates. The 2nm and 5nm gold particles must be enhanced with the Blotting Silver Enhancing Kit.
Gold Conjugates Labelling
a) Light Microscopy Gold particles cannot easily be seen in the light microscope by bright field viewing. They must thus be silver enhanced for greatest visibility. Small gold particles will give the greatest number of gold labelling on the specimen and each particle can then be subsequently silver enhanced for maximum visibility of signal. A choice can be made between 2nm and 5nm gold conjugates. The 5nm conjugates are used for most standard purposes and are recommended for initial studies. For even greater labelling intensity the 2nm gold conjugates are preferred. In both cases the gold signal is silver enhanced to make it visible in the light microscope.
c) Scanning Electron Microscopy The resolution of the scanning electron microscope indicates that larger gold particles (e.g. >20nm) should be used for detection by back scattering electron signals. However, as mentioned above, a greater intensity of labelling is achieved with smaller gold particles, which can be subsequently silver enhanced with the LM/EM Silver Enhancing Kit. We would therefore recommend that a choice is made for SEM studies between 20 or 30nm gold conjugates for direct (un-enhanced) viewing, or 5nm conjugates which can be simply silver enhanced within minutes for observation at low magnifications. With the latter approach each gold particle grows spherically to approximately 30nm in 35 minutes. The reaction is simply stopped by washing in water.
Microarray
Different particle sizes are appropriate for different types of application as described below.
immunogold labelling in the EM or performing studies over a range of magnifications, 10nm gold conjugates are recommended.
Silver Colloid
A wide range of particle sizes can be conjugated to proteins and macromolecules. In principle smaller gold particles produce a higher labelling intensity on the specimen. This is because of the reduced steric hindrance to antigen detection. Typically an antibody of 160,000 Daltons molecular weight will have a linear dimension of 8nm. Thus a 2nm gold particle, attached to the Fc region will hardly impede the antibody activity. A 20nm particle. however, while being more visible, will produce a greater steric hindrance by its proximity to the antigen binding region of the antibody. In addition the increased charge repulsion between larger particles reduces the number of labelled antibodies gaining access to the target antigen.
Gold Colloid
How to Choose the Gold Particle Size
Ordering
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Distribution
b) Transmission Electron Microscopy For electron microscopy any particle size may be employed. For low magnification work larger particles (e.g. 15 to 30nm) are more easily seen. For high magnification studies the smaller particles (e.g. 5 to 10nm) are preferred. Again, smaller particles give higher labelling intensity and may be subsequently silver enhanced on the section to produce larger particles with this high labelling intensity. For those just beginning
18
About BBI Gold Colloid
EM Grade Products
Silver Colloid
Gold labels are the most satisfactory method of labelling antigens for visualisation in the electron microscope. Since their introduction in 1971 there has been a vast growth in the number of applications in animal and plant biology as well as microbiology1. The main advantages of gold labelling lie in the high contrast and unequivocal nature of the label, the range of particle sizes suitable for different magnifications, the extreme sensitivity, and the stability of the signal.
Microarray
Features of BBI EM Grade Immunogold Conjugates Detailed datasheets available for download for all BBI gold reagents giving individual specifications for each product.
High OD
High Sensitivity Conjugates with particle sizes upto 20nm will detect 10pg of specific protein with each gold conjugate in immunoblotting tests (with silver enhancement).
Narrow Particle Size Distribution The low coefficient of variation of the gold particles for each conjugate allows multiple labelling to be achieved for several antigens on the same specimen. Long Shelf Life BBI Gold Conjugates are stable at 2-8°C for 12 months from date of manufacture. If the conjugate is stored frozen it will remain stable for longer. Conjugates stored frozen may be usable beyond their expiry date, but it is advisable to test sensitivity by immunoblotting first.
Which Gold Conjugate? The appropriate gold conjugate may be selected according to the criteria described in How to Choose a Gold Conjugate. The primary antibody determines the choice of secondary gold conjugate. F(abâ&#x20AC;&#x2122;) fragment gold conjugates may be appropriate where background staining is high.
Which Particle Size?
Gold Conjugates
Low Clustering Each gold conjugate has greater than 85% singlets. Quantitation is possible by counting particle density. Micrographs form attractive publications.
Labelling
Low Cross Reactivity All BBI antibodies are affinity purified and of the highest quality. Cross reactivity with other species is minimal. Conjugates are further absorbed against specific serum proteins where indicated.
Ordering
High Concentration Optical densities of gold conjugates at 520nm wavelength are measured as 3.0 for 5 and 10nm, 4.0 for 15 and 20nm or 5.0 for 30nm or 40nm EM Grade gold conjugates. This allows them to be used in a highly diluted form.
Distribution
High Resolution Because of the discrete nature of the gold particle and the close adsorption of anti-body/binding protein to the surface, antigens are localised with very high resolution.
19
For selection of particle size it is recommended that this is determined by the magnification to be used in the EM as in the table. For the highest labelling intensity, however, the smaller gold particles are preferred. For 2nm and 5nm gold conjugates, a combination of gold labelling with silver enhancing will yield larger size particles with high labelling intensity. For those researchers beginning with immunogold labelling we recommend 10nm gold particle size for observation at a range of magnifications. Please feel welcome to contact us for discussion of your application at info@buybbi.com
Types of EM Specimens Specimens may be prepared for labelling and examination in the electron microscope in a number of ways: a) Post Embedding Labelling of Sections This is the most widely used technique for labelling antigens for EM examination. Here the tissue is fixed and embedded and incubations performed on sections. Only the surface of the section is labelled. Ultrathin cryosections may be included in this category.
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About BBI
EM Grade Products Scanning Electron Microscopy (SEM) Large specimen areas may be examined by the SEM. Specimen surfaces are labelled by a pre-embedding method (b) and then prepared for SEM examination. Backscattered electrons provide an image of the gold particles on the specimen surface. Large gold particles (>20nm) may be imaged in the SEM with good resolution while the specimen surface is viewed with secondary electron emission. Alternatively, for higher labelling intensity, smaller (e.g. 5nm) gold conjugates are used and subsequently silver enhanced.
Methods of Use of EM Gold Conjugates
e) Freeze Fracture Freeze fracture is suitable for labelling cell membranes and for examination by thin sections or with replicas. With this technique it is possible to label plasma and intracellular membranes on isolated cells or within tissues.
Reference
Detailed instructions for use of all BBI gold conjugates and silver reagents are given in the Technical Information Booklet.
High OD
1 ‘Colloidal Gold& Principles, Methods and Applications”, Vols. 13, MA Hayat (Ed), Academic Press, 1989-1995*
Gold Conjugates
Primary electron beam
Microarray
d) Replica Technique Cell surface antigens on cultured cells may be examined with this method. Cells are fixed and labelled before being replicated for study by EM.
Silver Colloid
c) Negative Stain Method This is the most suitable method for identification of antigens on micro-organisms dried onto an EM grid.
Gold Colloid
b) Pre Embedding Labelling of Tissues and Cells In this case tissues (e.g. cell cultures or cells in suspension) are labelled by incubation, usually after fixation, and before embedding. This method is particularly appropriate for the identification of antigenic sites on membranes of both isolated cells and micro organisms.
Backscatter electron detector SEM display
Labelling
Secondary electron detector
Ordering
Gold particles +– Silver enhancemen nt Cell surface
Distribution
Detection of surface antigens by immunolabelling and scanning electron microscopy.
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20
About BBI Gold Colloid
Gold Conjugates
Silver Colloid
A colloidal gold conjugate consists of a suspension of gold particles coated with a selected protein or macromolecule (such as an antibody). BBI has a range of EM grade products that combine different antibodies on the range of particles predominantly 2, 5, 10, 15, 20, 30 & 40nm. BBI’s gold conjugates provide the following benefits.
Distribution
Ordering
Labelling
Gold Conjugates
High OD
Microarray
• Binding to BBI’s superior quality gold colloid ensures a very low level of clustering while maintaining high level stability and sensitivity • Only highest quality antibodies and proteins are used, meaning consistently reliable products • BBI’s gold conjugates average 95% singlet particle scommercially important reactions
21
Product Code
Size
EM. BSA5
5nm
EM. BSA10
Product
0.25ml
EM and all Conjugates
1ml
£75.00
£224.00 £2 224 4.00 0
EM. BSA Conjugates
£53.00
£149.00 £149 9.00 0
Microarray Conjugates
£121.00
£424.00 0
(except EM. BSA)
Conjugate Key BSA CGC GAF GAG GAM GAR GAT GFAR GMHL PAG RAG STP
Bovine Serum Albumin Cationic Colloidal Gold Goat anti Mouse IgG & IgM Goat anti Guinea Pig IgG Goat anti Mouse IgG Fc (Gamma) Specific Goat anti Rabbit IgG Goat anti Rat IgG Goat F(ab)’2 anti Rabbit IgG Goat anti Mouse IgG (H+L) Protein A Rabbit anti Goat IgG Streptavidin
Product Code
Size
Product Code
Size
Best Seller
EM. GAR5
5nm
EM. PAG5
5nm
10nm
Best Seller
EM. GAR10
10nm
EM. PAG10
10nm
EM. CGC5
5nm
Best Seller
EM. GAR15
15nm
EM. PAG15
15nm
EM. CGC10
10nm
EM. GAR20
20nm
EM. PAG20
20nm
EM. CGC15
15nm
EM. GAR30
30nm
EM. RAG5
5nm
EM. CGC20
20nm
EM. GAR40
40nm
EM. RAG10
10nm
EM. GAF10
10nm
EM. GAT10
10nm
EM. RAG15
15nm
EM. GAG5
5nm
EM. GFAR5
5nm
EM. RAG20
20nm
EM. GAG10
10nm
EM. GFAR10
10nm
EM. STP2
2nm
EM. GAM2
2nm
Best Seller
EM. GMHL5
5nm
EM. STP5
5nm
EM. GAM5
5nm
Best Seller
EM. GMHL10
10nm
EM. STP10
10nm
EM. GAM10
10nm
Best Seller
EM. GMHL15
15nm
EM.STP15
15nm
EM. GAR2
2nm
EM. GMHL20
20nm
EM.STP20
20nm
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About BBI
How to choose a Gold Conjugate
Gold Colloid
Technical Assistance
Silver Colloid
In addition to the choice of antibodies and particle size, the selection of the correct gold conjugate depends on the correct use of protocols for specimen preparation and incubation. At BBInternational we are happy to provide assistance in these methods and will gladly help in the choice of the correct gold conjugate for any specific application.
Microarray
Please contact our Technical Support team on +44 (0) 29 2074 7232 or e-mail us on info@ bbigold.com with your enquiry. In addition our web site www.buybbi.com has a FAQs section which includes the most common queries we have experienced.
High OD Gold Conjugates Labelling Ordering Distribution
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22
About BBI Gold Colloid
GoldLink
Silver Colloid
GoldLink™ kits are designed for early stage R&D, where users need to quickly explore the viability of a range of antibodies, before the conjugate is optimised on a larger scale. Users can now easily screen their antibodies of choice in-house in a cost effective way, before utilising BBI’s Custom Conjugation service to progress their development. This will streamline R&D time and ensure efficient use of budget in the early process of establishing which antibodies are right for the product.
The GoldLink™ Colloid in the kits is supplied as a freeze dried mixture.
Dilute Antibody
Mix with reaction buffer
Add to freeze dried BBI Gold Colloid Microarray
Features and Benefits
High OD
· BBI’s gold colloid · Quick and easy process for antibody conjugation – 15 minutes · No antibody pre-treatment required · Single approach to antibody screening · Minimal user knowledge or experience required · Conjugate stability assured · Minimal antibody usage · Minimal antibody waste
Gold Conjugates
Background and Procedure
Labelling
The kits, containing BBI’s unique recipe gold colloid, are stable to high salt buffers and are pre-activated for antibody labelling. The antibodies are attached to the functionalised gold by covalent conjugation through the lysine residues, which produce a stable, non-reversible linkage. No antibody pre-treatment is required. A single reaction buffer is used for conjugation of all antibodies. The process requires minimal antibody and does not need a purification step, meaning time and cost savings.
Quench the reaction
Finished Conjugate
Sensitivity The level of conjugate sensitivity achieved through the use of a GoldLink™ kit is suitable for antibody screening during initial R&D (see graph below). For customers who want to achieve a higher level of sensitivity and/ or a fully optimised conjugate, BBI also offers a Custom Conjugate service. BBI will take your antibody of choice and using our long established, proven processes, optimise your conjugate, and then freeze your specific recipe before putting a commercial supply plan in place.
Distribution
Ordering
Antibodies or proteins are covalently attached to ultrastable BBI Goldlink Colloid at a very high OD, quickly and easily (see Figure 1 below). The hands-on time for the kit procedure is just 2 minutes and the conjugate is ready to use in 15 minutes. The Goldlink Colloid has a protective surface coat that can withstand the most extreme conditions (e.g. 1M NaCl or 2.5M NaOH at 70oC for >1 hour).
23
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About BBI
GoldLink
Gold Colloid
Loading Comparison - BBI GoldLink™Kit vs. BBI Custom Conjugation
Silver Colloid Microarray High OD
Purchase a Kit The following kits are available.
Volume of Finished Conjugate
Price
Reactions
Gold Particle Size
GLK3.40
3
4 40nm 0n
50 50ul 20OD
£ £125 12 25
GLK3.40
10
4 40nm 0n
50ul 50 20OD
£ £315 31 15
GLK3.40
1
40nm 40n
1 1ml 20OD
£ £315 31 15
Gold Conjugates
Product Code
Labelling Ordering Distribution
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24
About BBI Gold Colloid
Getting the best from Immunogold Labelling
Silver Colloid
Detailed technical instructions for the correct use of all BBInternational research products can be found online. Each product has a corresponding datasheet available to download from www.buybbi.com that provides information on specification and performance.
Microarray
The primary aim of all immunolabelling procedures is to obtain a maximum specific signal with a minimum non specific background. This is true for EM sections, LM sections, and immunoblots on membranes. The results obtained in any immunolabelling procedure are limited mainly by the specimen preparation and incubation procedures. References given here describe a number of optimum conditions and protocols for immunolabelling in LM, EM and Blotting applications.
Specimen Preparation
Gold Conjugates
High OD
Correct specimen preparation procedures are absolutely crucial for optimum labelling of antigens in cells and tissues. Methods commonly used for ultrastructural preservation in the EM or morphological preservation in the LM must usually be modified to ensure that antigens are not only retained but also available for labelling. This often involves compromise with the structure but careful selection of preparation methods can yield excellent combinations of structural detail and immunochemical labelling.
Distribution
Ordering
Labelling
For LM and EM studies cells and tissue sections may be studied by pre-embedding, post embedding, or cryotechniques. In addition whole cells may be
25
examined as cytospins, cell smears, cells in culture, and cells in suspension 1,2,3,4. It should be recognised that each step of the preparation procedure is likely to incur some antigenic loss by extraction or alteration or masking. Cumulatively these losses may greatly reduce the overall labelling unless each step is optimised. This can usually only be achieved empirically but some guidelines are described below. a) Fixation Cells and tissues may be fixed for subsequent examination or may sometimes be labelled in the unfixed state. Fixation of the tissue must also preserve antigenicity without compromise to structural characteristics. Fixatives either denature proteins by coagulation (e.g. acetone or methanol) or by forming additive cross linked compounds (e.g. aldehydes), or both. The resulting complexes inevitably differ from the unfixed proteins in both their chemical and antigenic profiles. Each tissue requires its own fixation protocol. For example, too much cross linking in a tissue with high protein density may mask many antigens. On the other hand a loose tissue with low protein content may disintegrate without adequate fixation and antigens may simply be washed out. According to the antibodies employed for antigen detection, it may not be necessary to completely preserve the protein under investigation if at least the specific antigen is conserved. The types of fixatives employed are shown in the table below.
Types of Fixatives
Structure
Antigen Preservation
Suitability
Additive (cross linking) Formaldehyde Glutaraldehyde
++ +++
+ + ++ +
T isssue es Tissues T Tissues isssue es
Coagulative Acetone Methanol
+ +
+ +
C Cells ells C Cells ells
Mixed Formal Acetone Picric Acid
++ ++
+ +
C ells Cells T isssuess Tissues
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About BBI
Whatever method of fixation is selected it must serve the dual function of retaining the essential structural and antigenic components of the tissue without introducing any material which may interfere with the labelling.
In some cases the introduction of heavy metals such as osmium or uranium into the tissue may cause increased non specific labelling and must be treated with caution.
Cross Linking
Hydrophilicity
Structure/ Stability
Labelling
Types of Resin
Gold Conjugates
c) Embedding Tissues may be embedded in paraffin wax for LM, and resin for LM or EM studies. In either case the embedding should allow good preservation of the antigens without sacrifice of structural information. Generally there are two types of resin, epoxy resins which have an aromatic structure and are strongly cross linked, and acrylic resins which have lower cross linking. Epoxy resins are hydrophobic whereas acrylic resins may be hydrophobic or hydrophilic. The best structural preservation and stability is provided by epoxy resins while the best immunolabelling is usually achieved with acrylic resins. This is because acrylic resins cut in such a way as to reveal the proteins at the section surface and they also wet more easily, thus giving greater accessbility to the antibodies during subsequent incubations. Resins for EM and LM preparation are shown in the table below.
High OD
Post fixation for electron microscopy has mostly involved the use of osmium tetroxide in order to preserve membrane components and provide image contrast. The introduction of osmium into tissue is not always desirable, however, and more recently tannic acid has been suggested as an alternativeâ&#x20AC;&#x2122;.
Microarray
b) Washing Thorough washing of the tissues following fixation is extremely important. It may be necessary to wash for at least as long as the tissue has been fixed in order to remove excess aldehyde or other fixation residues which may cause non specific labelling. In some cases quenching the tissue with ammonium chloride is performed to neutralise aldehyde groups. The wash is best performed in a buffer having a tonicity similar to the natural tissue state.
Silver Colloid
For many tissues the best compromise is a mixture of formaldehyde (e.g. 2-4%) for rapid stabilising with low cross linking, and weak glutaraidehyde (e.g. 0.1%) for greater structural preservation. For cytological investigation a precipitating or coagulating fix such as acetone or methanol may be preferred. Formal acetone has also been commonly used for fixing cell preparations. In some cases for cell studies simple air drying may allow enough antigenic preservation for immunolabelling.
Gold Colloid
Getting the best from Immunogold Labelling
Antigen Preservation/ Access
Epoxy High
H Hydrophobic ydrop pho
++ +++ ++
+
Epon
High
H ydrop pho Hydrophobic
++ ++ +++
+
UNICRYLTM
-
Hydrophilic Hydrop phili
++ +
+ +++ ++ +
Lowicryl
-
H ydrop phili Hydrophilic
++ +
+ + ++
LR White
-
H ydrop phili Hydrophilic
++ +
+ + ++
LR Gold
-
H Hydrophilic ydrop phili
++ +
+ ++ +
Methacrylate
-
+/ +//- H Hydrophilic yd dro
++ +
+ ++ +
Ordering
Araldite
Acrylic Distribution
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26
About BBI Gold Colloid
Getting the best from Immunogold Labelling
Microarray
Silver Colloid
For the optimum results a compromise must be reached and the usual choice is of a polar (hydrophilic) resin with moderate cross linking. A popular acrylic resin formulation is given by UNICRYLTM, which gives excellent immunolabelling characteristics together with a high degree of stability and structural preservation. UNICRYLTM, in common with many other acrylic resins, also allows the embedding procedure to be performed at low temperatures. This ensures that vital components are not extracted during dehydration and that no excessive temperature rise occurs during polymerisation which may damage the tissue antigens,
Ordering
Labelling
Gold Conjugates
High OD
d) Blotting For proteins blotted onto membranes by Western blotting or dot blotting it is important to ensure complete transfer of the proteins to the membrane. Membranes must then be blocked with neutral proteins or surfactants (e.g. BSA or Tween20ÂŽ) to fill unoccupied sites and prevent non specific background labelling. The membranes are then incubated in a similar fashion to tissue sections. The complete protein band/spot pattern may first be revealed by immersing the membrane in a solution of PROTOGOLDÂŽ which stains all proteins with colloidal gold, yielding a red stain. This gives an indication of the location of all protein bands. A second identical membrane may then be incubated with the appropriate specific antibodies for identification of specific proteins immobilised on the membrane. An incubation of the membrane with gold labelled second antibodies will reveal the presence of specific protein bands by a visible red colour where the gold particles accumulate. In order to achieve the best signal with the least background thorough washing is important after each step.
Incubations
apply for all types of tissues, whether for EM or LM, and for immunoblots on membranes. a) Primary antibody The primary antibody should be of high titre and of the highest specific purity to allow the greatest possible dilution. Cross reactivity must be low, especially with the sample tissue. A low quality primary antibody is the greatest cause of low specific signal and high background during the labelling procedure. The buffer composition and pH is important for the incubations. A normal TBS or PBS buffer is usually chosen with suitable additions to maintain low background. The specimen must be washed thoroughly with buffer between incubations. (b) Secondary antibody A high quality second antibody is essential to label the primary specifically and with low background. BBInternational gold conjugates are affinity purified and of the highest quality. They are provided suspended in Tris or Phosphate Buffered Saline for microscopical or immunoblotting use, with sodium azide preservative and are stable for years under the correct storage conditions. This is because our gold conjugates contain no free antibody, all antibodies being securely adsorbed at the surface of the gold. In addition the very high proportion of singlets ensures that clusters will not form in time during storage. Their high titre and purity means that for incubations they may be used highly diluted (typically 1/100 to 1/400) in the typical incubating buffer shown below, so reducing non specific background whilst achieving a high signal intensity. Even at high dilutions (1/1000 or more) the conjugates are extremely stable and may be left for long term incubations.
Distribution
In order to achieve the highest possible specific signal with the lowest possible non specific background it is important to be aware of all the factors in both specimen preparation and in subsequent incubations which can affect these results. The figure above indicates some of the most common sources of non specific background that can be identified by the use of controls and successfully eliminated. These factors
27
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About BBI
Serum cannot be used in conjunction with Protein A, Protein G or Protein AG gold conjugates since these bind IgG molecules.
Silver Colloid
Select serum corresponding to the host of the gold labelled antibody. In this example goat is selected because the conjugate being used is Goat anti-Rabbit. e) Controls In order to determine if a signal is genuine and to differentiate it from background labelling, both positive and negative controls must be used. These are simple to perform and should always be included in any staining protocol. Negative controls would typically include the following:
Microarray High OD
• Omit the primary antibody • Use a non specific primary antibody of the same species
Gold Conjugates
(c) Dilutions When establishing a new protocol it is necessary to determine the optimum concentrations of both the primary and the gold labelled secondary antibodies. This is done by first incubating separate sections with various dilutions of the primary antibody over an appropriate range of values, e.g. 1/100 to 1/10,000. The various primary incubations are then followed by second incubations, using a constant dilution such as 1/100 of the gold labelled secondary antibody. The dilution of primary antibody giving the optimum signal and background is thus determined. The procedure is then repeated, this time using the determined dilution of the primary as a constant, and incubating with a selected range of dilution values of the gold conjugate, e.g. 1/10 to 1/400. Observation of the second set of results will indicate the choice of dilution of conjugate that should be used in further experiments. With both primary and secondary incubations a greater sensitivity may also be achieved by agitating the sample or flushing the reagents, so bringing fresh solution continuously to the target proteins at the tissue surface. If the antibody solutions are tolerant the incubations may also be performed at slightly elevated temperatures (up to 37°C).
Gold Colloid
Getting the best from Immunogold Labelling
• Absorb the primary antibody with antigen before incubation • Use a non specific second antibody
Ordering
For LM, EM and Blotting applications the correct use of silver enhancing procedures is also important to maximise the signal and to avoid non specific background.
Distribution
A typical incubating buffer suitable for both LM and EM incubations, which provides the components for eliminating the above sources of background may be as shown below. This buffer may be used for each step of the incubation protocol, followed by washing in water after the final step.
These negative controls will help identify the source of any non specific background. A good positive control using a specimen with high antigen content will test the whole labelling system. Full instructions for the use of controls are given in our Technical Instruction Booklet.
Labelling
d) Blocking Non specific reactive sites on tissues and cell surfaces as well as unoccupied sites within blotting membranes may need blocking before antibodies are applied to the specimens. The causes of non specific labelling may arise from sources shown in the table. Each background source will need its own blocking procedure either before or during the antibody incubations as shown in the table and as described in the Technical Information section. BBInternational can supply blocking reagents.
The technical Information online at www.buybbi.com gives full details of basic protocols, dilutions, buffers, blocking reagents, controls, trouble shooting and recommended reagents for many applications to help achieve the best from immunogold labelling.
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28
About BBI Gold Colloid
Gold Stains PROTOGOLDTM TM
Silver Colloid
PROTOGOLD colloidal gold sol has been specially developed to give intense staining of proteins blotted onto membranes by dot blots or by electrophoretic gels (Western blotting). The gold particles are negatively charged and bind selectively to the blotted proteins with very low background absorption on the membrane. The specific staining of proteins occurs primarily by hydrophobic and ionic interactions.
Microarray
The proteins stain dark red through the accumulation of gold particles on the membrane and the method produces a sensitivity greatly in excess of Coomassie blue or silver staining in gels with no fading of the signal. When used in conjunction with the BBI BL Silver Enhancing Kit a further enhancement of the signal by 10 to 100x is achieved.
High OD
Why Stain Membrane Blots?
Gold Conjugates
Staining of proteins on gels has been an established technique for many years. For the identification of individual proteins in a gel, however, and for permanent storage, proteins must be transferred by blotting to a membrane such as nitrocellulose or PVDF. Individual protein bands can then be identified by immunoblotting using specific primary antibodies followed by BBI secondary gold labelled antibodies.
Labelling
Three significant problems arise when trying to compare the total protein stain on a gel with an individual specific protein band on a membrane.
Ordering
a) Transfer of the proteins from gel to membrane may not be the same for each protein band since the very nature of the electrophoresis method differentiates proteins by their size, charge and mobility.
Distribution
b) Distortion between the gel and the membrane may occur so that matching proteins on the gel with individual proteins on the membrane may be difficult.
For these reasons it is sensible to compare individual specific proteins stained by immunoblotting on a membrane with total proteins stained on a duplicate membrane with PROTOGOLDTM. The method is extremely easy and results are very rapid. By combining total protein staining with PROTOGOLDTM and specific immunostaining of individual protein bands with an enzyme labelled antibody (e.g. alkaline phosphatase) both signals can be achieved on the same membrane.
Features of PROTOGOLDTM Fast Results Proteins are stained in minutes. High Sensitivity Less than 1pg of protein is detectable (with silver enhancing). High Resolution A crisp image of separated protein bands is produced on the membrane. Easy to Use The single solution is used straight from the bottle. Economical PROTOGOLDTM may be re-used on successive blots until the gold is exhausted. Quantitative Determination Proteins are detectable from solution below 1ng/ml. Cannot Overstain The membrane may be left in solution. No destaining is necessary. Long Shelf Life PROTOGOLDTM is stable at 2-8°C for until its expiry date. This product cannot be frozen.
c) Handling and permanent storage of a gel, together with quantitative estimation of protein bands is difficult.
29
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About BBI
Gold Stains
Gold Colloid
Further Technical Help
Method of Use
Further advice on using PROTOGOLDTM in the technical instruction booklet.
TM
The application of PROTOGOLD to proteins blotted on a membrane is very straightforward.
Silver Colloid
PROTOGOLD® is a regestered trademark of BBInternational Ltd
a) Incubate the blotted membrane with 0.3% Tween20 detergent to block unoccupied sites and to eliminate background.
Tween20® is a regestered trademark of Atlas Chemical Industries
b) Wash in deionised water to remove buffer salts
Microarray
c) Incubate the membrane with PROTOGOLDTM in a tray with continuous gentle agitation. After 2 to 3 minutes, proteins begin to appear stained light red. After 10 minutes the red colour deepens on the strongest bands. Optimal staining occurs after 2 hours. The membrane may be incubated indefinitely without overstaining. There is no need to destain.
High OD Gold Conjugates
If necessary the membrane can then be silver enhanced to give a further 10 to 100 times signal intensity with the BBI BL Silver Enhancing Kit. d) Wash the membrane in water and dry.
Quantitation of Proteins from Solution
Price
PROTOGOLD® Kit
500ml
PRO500
£1 £107.00 07 7.00 0
Tween20® detergent
10ml
T20
£9 £9.00 9.00 9 0
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Distribution
Product Code
Ordering
Unit Size
Product
Labelling
Individual proteins in solution can easily be quantified by direct dot blotting onto nitrocellulose and staining with PROTOGOLDTM. The resulting stain is compared with standards or a titrated series of protein blots stained simultaneously. Less than 1ng/ml of protein can be detected by this method.
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About BBI Microarray
Silver Colloid
Gold Colloid
Silver Enhancing Products The BBI Silver Enhancing kits provide simple to use and sensitive systems for the amplification of immunogold labels in light microscopy, electron microscopy and blotting applications. Enhancement occurs during the reduction of silver from one solution (the Enhancer) by another (the Initiator) in the presence of gold particles. This reduction causes silver to build up on the surface of the gold particles that are attached to antibodies, proteins or other macromolecules on the target specimen. Enhancement is rapid but easily controllable within a comfortable time span (minutes). Amplification of the gold signal by 10 to 100 times is readily achieved. The reaction is insensitive to light, is simply stopped by washing in water, and needs no fixing.
slip in place to allow high magnification viewing, even with oil immersion. The reaction is stopped by simply washing in water. It may be continued if necessary with fresh solutions.
BBI provide two silver enhancing kits for different applications:
Epipolarised Light
High OD
• LM/EM Silver Enhancing Kit - for light and electron microscopy
Gold Conjugates
• Blotting Silver Enhancing Kit - for blotting applications • Light Microscopy
Ordering
Labelling
Specimens may be immunogold labelled sections of tissue, whole cells, smears, chromosomes, etc mounted on glass slides, cell monolayers or tissue slices in culture. Small gold particles are not visible in the LM because of the limit of resolution (>200nm) of the microscope. After the immunogold labelling the specimen is washed in water and simply enhanced in a single step with the solutions provided. The final result is an intense brown/black stain at the site of the gold label.
Distribution
5nm immunogold labels give excellent results with silver enhancement, 2 nm gold labels being of particular value when penetration of cells is required. Adequate amplification is usually obtained within a period of 5/10 minutes and may be monitored on the microscope during the reaction. The enhancement is performed by mixing together one drop of each solution and applying to the slide. The reaction can be performed with a cover
31
The LM/EM Silver Enhancing Kit is sufficient for at least 300 incubations on glass slides. After washing, the slides may be counterstained and mounted as normal. Because of the discrete nature of the growth of silver on the gold particles, no diffusion of the signal occurs. The silver stain produces a permanent, non-fading label of sharp resolution and high contrast in bright field viewing. BIOMOUNT may be used to avoid leaching of the silver signal that may occur with other types of mountant.
In conjunction with epipolarised light the gold/silver enhancement also gives a brilliant image of the signal against a dark background in the light microscope. In this arrangement, the epipolarised light source (usually UV) is reflected by the perfectly spherical silver ‘mirrors’ formed by enhancement of the gold particles. Sensitivity is increased by 10 times in comparison with normal bright field viewing. This means that lower levels of gold are needed for visualisation, thus producing a much improved peak to background signal. Most modern microscopes can easily and inexpensively be equipped with an epipolarising light attachment by the manufacturer.
Electron Microscopy Any gold label on an electron microscope specimen may also be effectively enhanced with the LM/EM Silver Enhancing Kit. Silver enhancing for electron microscopy is especially useful where a low labelling intensity, due to very low levels of tissue antigen, requires that 1nm or 5nm gold conjugates must be used. With these small particles the best possible labelling intensity is achieved but the particles may not be so easily visualised, especially in electron microscopes of limited magnifying power. By floating the EM grid carrying the ultrathin specimen on a drop of enhancing solutions for a few minutes the particles become large enough to see at much lower magnifications. It is quite common to observe these particles, which can be grown to 50nm or more, at only 10,000 times magnification or lower. The immunolabelling can therefore be observed within a
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About BBI
Silver Enhancing Products
The Blotting Silver Enhancing Kit is provided as two separate solutions. Equal volumes (e.g. 10ml) of each solution are mixed before use. After gold staining, the membrane is washed in water and then simply immersed in the mixed solutions for the appropriate time. The reaction is stopped by washing in water.
For SEM studies any gold particle can be used in the immunogold reaction and these particles grown to a size (e.g. 50nm) which is convenient to view by scanning using backscattered electrons or X-ray mapping. The use of small gold particles, e.g. 5nm, with subsequent silver enhancing is a preferred approach to labelling SEM specimens compared to the use of larger gold particles for direct viewing because of the higher labelling intensity.
Microarray
Scanning Electron Microscopy
PROTOGOLD®, is a registered trademark of BBInternational Ltd (BBI)
Blotting Applications
These strips allow the sensitivity and stability of the kit to be monitored over a period of time. They may also be used in parallel with each enhancing reaction. Packs of 10 Test Strips are available separately.
The Blotting Silver Enhancing kit is formulated to produce an intense, sharp, black signal from gold labels on specimens such as proteins immobilised on
No. of Reactions
Product Code
Gold Conjugates
Unit Size
High OD
Test Strips
Product
Silver Colloid
membranes. It is therefore ideal for enhancing immunogold or total gold stains from Western, Southern, Northern, dot blots, using the PROTOGOLD® and GENOGOLDTM products and the blotting grade immunogold reagents. With this system, immunogold/silver staining provides a safe, sensitive, rapid and economical alternative to radiolabelling procedures.
Gold Colloid
much wider specimen area than at high magnification. The gold particles grow in a perfectly spherical manner but when adjacent particles touch they fuse to form elliptical shapes. The technique is so simple that specimens may be enhanced repeatedly and observed in the microscope until the required particle size is achieved.
Price
2 x 15ml
>300
SEK SEKL15 KL15
£78.00 £78.0 00
BL Silver Enhancing Kit
2 x 250ml
20 to 30 blots
SEK SEKB250 KB250 0
£107.00 £107 7.00 0
Test Strips
Pack of 10
10
SETS SETS10 S10
£20.00 £20.0 00
Labelling
LM/EM Silver Enhancing Kit
Ordering
Further technical and procedural help on LM/EM silver kits and BL silver kits, please contact our technical support team on +44 (0) 29 2074 7232 or e-mail info@bbigold.com.
Distribution
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32
About BBI Gold Colloid
UNICRYLTM TUNICRYLTM is a unique acryllic resin whuch has been developed for universal use in both light and electron microscopy1. It has the following applications:
Silver Colloid
Light Microscopy Histology Histochemistry Immunocytochemistry Immunolabelling In Situ Hybridisation
Electron Microscopy Ultrastructure Cytochemistry Immunocytochemistry Immunolabelling In Situ Hybridisation
Microarray
UNICRYLTM provides optimum sectioning, labelling and staining qualities for studies in animal, plant and microbiological tissues
UNICRYLTM is unique
Labelling
Gold Conjugates
High OD
The advantages of UNICRYLTM for staining and labelling lie in both its preservation of tissue structures and its sectioning characteristics such that proteins, nucleic acids and macromolecules are revealed at the surface of the sections for subsequent incubations. The resin preserves these structures without chemically interacting or cross linking with them. UNICRYLTM is largely hydrophilic, allowing good access to polar (aqueous) solutions and exhibiting a low background staining or labelling from hydrophobic materials. It also minimises the denaturation of proteins, allowing true antigenic properties to be maintained. Normal counterstaining properties for both EM and LM studies are excellent due to the hydrophilicity of the resin and its homogeneous structure1,2,3,4,5.
Storage UNICRYLTM is stable at -20°C until its expiry date. Each UNICRYLTM kit comprises 250ml of resin, embedding vials, plastic gloves and a disposable bottle. A detailed instruction booklet explaining methods for specimen preparation and polymerisation at high and low temperatures is provided with each UNICRYLTM kit. The UNICRYLTM STAINING KIT is stable at 2-8°C, until its expiry date.
Safety UNICRYLTM resin contains methacrylates, styrene and benzoyl peroxide. Please read the UNICRYLTM MSDS for safe handling and disposal information.
Acknowledgements UNICRYLTM resin was originally developed by the research group of Dr C Scala at the Institute of Clinical Electron Microscopy, University of Bologna, Italy. In the papers of Scala et all, the UNICRYLTM resin is referred to as Bioacryl, its original name. The staining protocols follow modifications of the method of Scala et al.
Ordering Information Product UNICRYL® Resin Kit
UNICRYLTM is easy to use
Distribution
Ordering
The resin is provided as a single solution and is used in a similar way to other acrylic resins for embedding tissues. Because it is a single solution no mixing is required. The resin has a long shelf life if stored in the cold. It is miscible with alcohols and has a low viscosity even down to –50°C. UNICRYLTM can be polymerised by heat or by UV irradiation at lower temperatures. Tissue pieces are processed in single capped 1ml eppendorf tubes, BEEM capsules or gelatin capsules by simple pipetting of solutions into the tubes. Cells in culture may be processed in situ in vials enclosed during polymerisation.
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Unit Size
Product Code
250ml
BA250
Price £1 £146.00 146 6 00 6.00
About BBI
Ancilliary Products For light microscopy and electron microscopy the choice of specimen preparation is critical for the preservation of antigens in the sample. Of greatest importance in the preparation schedules are the specimen fixation and embedding. The protocol must satisfy the requirements for preservation of structural integrity and antigenicity.
BIOMOUNTTM reduces fading of immunogold/silver signals in sections on glass slides. It is suitable for both resin and wax embedded tissue sections.
BIOMOUNTTM is miscible with xylene and may be applied to sections on slides in the normal manner following dehydration and immersion in xylene. Labelling will retain its intensity and contrast following the use of BIOMOUNTTM. Sections that have been counterstained with normal stains or with the UNICRYLTM STAINING KIT will also retain their colour intensity.
Product
Unit Size Product Code
Gold Conjugates
Price
BIOBONDTM Tissue Adhesive
20ml
BB20
£36.00 £3 36..0 00 0
BIOMOUNTTM Tissue Mounting Medium
100ml
BM100
£25.00 £2 25..0 00 0
Ordering Distribution
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Labelling
BBI now offers a special slide coating solution, BIOBONDTM, which produces a very strong adhesion between the glass and the tissue section for subsequent incubations. BIOBONDTM coats the slide with a protective layer to minimise interaction of charged glass surface with reagents. This is also of particular importance for reproducibility of results because of the variations that occur between glass slides obtained from different sources and in different countries. It is particularly effective for use with severe incubating conditions such as those used in in situ hybridisation. BIOBONDTM is suitable for all kinds of tissue specimens including paraffin wax or resin sections, cell smears, cytospins or cryostat sections. BIOBONDTM is supplied in 20m1 unit volumes, sufficient to coat at least 1000 slides.
High OD
In addition the surface of glass slides is uneven and is activated by the silicon tetrahedral structure. This provides active sites for absorption of proteins or reactions with chemicals and reagents. It is therefore important to minimise this possibility by coating the surface with a material that is of low reactivity towards reagents.
Microarray
Having prepared the tissue specimen for immunolabelling it is then imperative to perform the incubations with a protocol designed to maximise the specific signal and minimise the background. Some incubation conditions may cause tissue sections to be removed from the glass slide. Typical tissue section adhesives such as poly-L-lysine, Elmer’s glue, chrome alum, etc are not suitable for use with immunogold labelling because of the increased background caused by attraction of gold particles to the adhesive on the slide.
Some mounting media oxidise rapidly on exposure to air, forming carboxyl groups. This may be especially so when sections have been cleared in a solution containing aldehyde groups. It is sometimes observed that the immunogold / silver stain fades after a few weeks, or even in a shorter time, from sections that have been mounted with these media under cover slips. The silver is still present, but has formed translucent silver carboxylate salts. Visibility can be retrieved by removing the cover slip and washing in xylene (xylol) and then immersing the slide in photographic developer, but this is a tedious procedure. BIOMOUNTTM prevents this fading because of its low oxidising properties.
Silver Colloid
BIOMOUNTTM Tissue Section Mounting Medium
Gold Colloid
BIOBONDTM Tissue Section Adehsive
34
About BBI Gold Colloid
Ancilliary Products Blocking Reagents
Silver Colloid
Non-specific labelling can occur on specimens during immunolabelling procedures. The source of this background labelling must be determined by the careful and systematic use of controls and eliminated for a proper analysis of the specimen. Background labelling can occur from a number of sources, either in the specimen or in the incubating solutions. In either case the background can be substantially reduced by the careful use of blocking reagents.
Product
Unit Size Product Code
Price
Tween 20®
10ml
T20
£9.00 £9 9.00 0
BSA (fatty acid free)
10g
BSA10G
£3 39.0 00 £39.00
Gelatin (fish 45%)
10ml
GEL10
£20.00 £2 20.0 00
Microarray
Tween 20® is a registered trade mark of Atlas Chemical Industries
Labelling
Gold Conjugates
High OD
• Background labelling from the specimen • Hydrophobic attraction of proteins or gold particles • Charge attraction from tissue components to antibodies or gold particles • Fixing of antibodies through excess aldehyde groups in tissue • Receptors in tissues for Fc components of antibodies • Excess sulphur in tissue or support • Excess lysine in tissue adhesive (for LM sections) • Background labelling from incubating solutions • Hydrophobic proteins (e.g. antibodies) • Charged proteins (e.g. pH, ionic concentration) • Charged gold particles • Gelatin in buffer
Distribution
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Most of these sources of background can easily be overcome by the addition of appropriate blocking reagents to the buffers used for incubating or by a separate blocking step before the primary antibody. Full details of the correct use of controls to identify the background source and the subsequent use of blocking reagents is given in the Conjugate Technical Information section.
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About BBI
How to Order BBI is a global supplier and has 25 years’ experience in the manufacture of superior quality gold nanoparticles and secondary gold probes for the Life Sciences and nanotechnology field. Our products are guaranteed for a high level of stability and consistency and our customers are supported by a team of technical support specialists and distributors in over 200 countries.
To ensure your privacy and safety when registering and ordering online all communications between your web browser and our server is encrypted using a secure socket layer (SSL) protocol.
Ordering Directly From BBI You can place an order quickly and securely by credit/ debit card using our online shopping cart.
By Telephone +44 (0) 29 20 74 7232
When you are done shopping, navigate to the checkout page using the top right menu. When placing your first order you will be asked to register for an online account. Registering for an account also gives you access to special offers, exclusive promotions, newsletters and new product information.
High OD
Alternatively if you do not have the ability to use a credit or debit card you may order direct from us. BBI accepts institutional purchase order from all over the world. You can place your order by contacting us:
To order securely online firstly select the desired currency at the top of the page (Prices listed are in GBP and in US dollars for international customers). Then choose the products of interest in the size, volume and quantity you require. Once selected, add your products to the shopping cart using the “Buy” button.
Microarray
Browse our products and services online or contact one of the friendly BBI team to find out how we can help you.
Silver Colloid
Online Ordering Instructions
Gold Colloid
Ordering Information
Shipping, Return & Replacement Policy Please review our shipping policy page on www.buybbi.com for more information.
Gold Conjugates
Fax your order to: +44 (0) 29 20 74 7242 E-mail your order to: info@buybbi.com
Labelling Ordering Distribution
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36
About BBI Gold Colloid
BBI Distributors Australia
Silver Colloid
Taylor Biomedical PTY Ltd 5/ 31 Howe Street Victoria Phone: +61280844762 E-Mail: office@taylorbiomedical.com.au
Pin-An-Nuo Technology (Beijing) Co., Ltd. Department shuiqingmuhua, zhongguancun, Haidian District, Beijing, 100190 Manager: Xinyue Wen QQ: 497465865 Email: bbi_bj@yahoo.cn
Labelling
Gold Conjugates
High OD
Microarray
Austria Christine Groepl Electronenmikroskopie Frauenhofnerstrasse 40 A-3430 Tulln Austria Phone: + 43 22 726 3177 Fax: + 43 22 726 31774 E-mail: christine.groepl.elmi@aon.at
Beijing LeBo Biotech Company Room 912,9f, Tongyuan Tower, No.95, Qinghe 3th Street, Haidian District, Beijing Phone: (86)-10-59564854 (86)-10-59564854 Fax: (86)10-62955993 E-mail: lab_china@163.com Web: www.lab-bio.com
Belgium
Czech Republic & Eastern Europe
TEBU-BIO NV Alexander Franckstraat 196 B-2530 Boechout Phone: + 03 454 00 66 Fax:+ 03 454 18 88 E-mail : ana.arraztio@tebu-bio.com Web: www.tebu-bio.com
BARIA s.r.o. Jižní 393 252 44 Psáry Czech Republic Phone/Fax: 00420 323 550 123 Email: nicolettek@baria.cz Web: www.baria.cz
Canada
Denmark, Finland and Norway
CEDARLANE 4410 Paletta Court Burlington ON L7L5R2 Canada Phone: 1-800-268-5058 Fax: 1-289-288-0020 E-mail: sales@cedarlanelabs.com Web: www.cedarlanelabs.com
Cytotech - Nota Bene Scientific ApS Nordre Strandvej 119F DK-3150 Hellebaek Denmark Phone: +45 70 23 20 60 Fax: +45 70 23 20 80 E-mail: info@nbs.dk Web: www.nbs.dk
Ordering
China
Distribution
Unisize Technology (Changzhou) Co., Ltd. No.801, Middle Road Chang Wu, Wujin District, Changzhou, Jiangsu Province Phone/Fax: +86-519-81195258 Email: umsphere@163.com Web: www.unisizetech.com
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About BBI
BBI Distributors TEBU-BIO SA 39, Rue de Houdan F-78612 Le Perray en Yvelines Cedex Phone: +01 30 46 39 00 Fax: +01 30 46 39 11 E-mail: ana.arraztio@tebu-bio.com Web: www.tebu-bio.com
Prodotti Gianni S.p.A. Via Quintiliano, 30 - 20138 Milano Italy Phone: +39 02 509 7 509 Fax: +39 02 509 7 276 E-mail: faresin@prodottigianni.com Web: www.ricerca.it
Silver Colloid
Italy
Gold Colloid
France
Japan Germany
High OD
Korea WOOMYOUNG INC 5F, KT Building, 734 Suseo-Dong Kangnam-Gu, Seoul 135-886 Korea Phone: +82-2-3412-6999 Fax: +82-2-417-2863 E-mail: woomyoung@empas.com Web: www.woomyoung.co.kr
Gold Conjugates
POLYSCIENCES EUROPE GmbH Handelstr 3 D 69214, Eppleheim Germany Phone: +49 6221 765 767 Fax: +49 6221 764 620 E-mail: info@polysciences.de Web: www.polysciences.com
Microarray
PLANO W PLANNET GmbH Ernst-Befort-Strasse 12 35578, Wetzlar Germany Phone: +49 6441 97 650 Fax: +49 6441 976 565 Email: jthorn@plano-em.de Web: www.plano-em.com
FUNAKOSHI Co. Ltd 9-7 Hongo 2-chome Bunkyo-ku Tokyo 113-0033 Japan Phone: +81 3 5684 1620 Fax: +81 3 5684 1775 E-mail: reagent@funakoshi.co.jp Web: www.funakoshi.co.jp
Hong Kong
Distribution
BIO-NET 26 Rachel Imenou Jerusalem 93228 Israel Phone: +972 2 567 1553 Fax: +972 2 563 1963 E-mail girasten@actcom.co.il
Synergy Scientific Sdn Bhd 29-2, Plaza Danau 2, Jalan 109F, Taman Danau Desa, 58100 Kuala Lumpur, Malaysia Phone: +603 79832632 Fax: +603 79832635 E-mail: alycia@synergyscientific.com.my
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Israel
Malaysia
Labelling
ADVANCED TECH. & IND. CO., LTD. UNIT B, 1/F, Cheong Shing Ind. Bldg., 17 Walnut St., Tai Kok Tsui, Kln, HONG KONG Phone: 23902293 Fax: 27898314 Email: sunny@advtechind.com
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38
About BBI Silver Colloid
Gold Colloid
BBI Distributors Netherlands
Sweden
SanverTECH bv Bevelandseweg 28 NL-1703-AZ Heerhugowaard The Netherlands Phone: +31 72 572 2100 Fax: +31 72 572 2133 E-mail: ana.arraztio@tebu-bio.com Web: www.tebu-bio.com
ANALYTICAL STANDARDS AB Metallvagen 5 S0-43533, Molnlycke Phone: +46 31 88 08 10 Fax: +46 31 88 08 86 E-mail: anastand@nordlab.se Web: www.nordlab.se
New Zealand Microarray
Taylor Biomedical PTY Ltd 5/ 31 Howe Street Victoria Phone: +61280844762 E-Mail: office@taylorbiomedical.com.au
High OD
Singapore & South East Asia
Gold Conjugates
Synergy Scientific Sdn Bhd 29-2, Plaza Danau 2, Jalan 109F, Taman Danau Desa, 58100 Kuala Lumpur Malaysia Phone: +603 79832632 Fax: +603 79832635 E-mail: alycia@synergyscientific.com.my
Spain
Ordering
Labelling
Abyntek Biopharma,S.L. Parque Tecnológico de Bizkaia Edificio 504 48160 Derio - Bizkaia Phone: (+34) 94 404 8080 Fax: (+34) 94 404 8081 E-Mail: jrivas@abyntek.com Web: www.abyntek.com
Switzerland CHEME BRUNSCHWIG AG Belchenstrasse 12 Postfach, CH 4009 Basel Switzerland Phone: +41 61 308 91 15 Fax: +41 61 308 91 19 E-mail: adm@brunschwig-ch.com Web: www.brunschwig-ch.com
Thailand & South East Asia Synergy Scientific Sdn Bhd 29-2, Plaza Danau 2, Jalan 109F, Taman Danau Desa, 58100 Kuala Lumpur Malaysia Phone: +603 79832632 Fax: +603 79832635 E-mail: alycia@synergyscientific.com.my
Taiwan Hygene Biotech Company Ltd ᾏ⛉⏕ᢏ᭷㝈බྖ 1F., No.10,Lane 4, Sanfu St., Wenshan District, Taipei City 116, Taiwan Phone: 886-2-2933-6382 Fax: 886-2-8663-1471 E-mail: michael.lai@hygene.com.tw Web: www.hygene.com.tw
Distribution
VITRO S.A. Apartado No. 1286 F.D. 28080 Madrid Spain Phone: +34 91 535 39 60 Fax: +34 91 535 27 80 E-mail: admvm@vitrosp.com Web: www.vitrosp.com
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About BBI
BBI Distributors
Gold Colloid Silver Colloid
LEVEL BIOTECHNOLOGY Inc 9F-3 no 21 Lane 169 Kong Kin Street His-Chih 221, Taipei Hsien Taiwan Phone: +886 2 2695 1252 Fax: +886 2 2695 7393 Web: www.level.com.tw
United Kingdom
Microarray
2B Scientific Ltd Cherwell Innovation Centre 77 Heyford Park Upper Heyford OX25 5HD Phone: +44(0) 1869 238033 Fax: +44(0) 1869 238034 Email: farjada@2BScientific.com Web: www.2BScientific.com
High OD Gold Conjugates
AGAR 66A Cambridge Road Stanstead Essex Phone: + 44 (0) 1279 813 519 Fax: + 44 (0)1279 815 106 E-mail: websales@agarscientific.com Web: www.agarscientific.com
USA Labelling
Ted Pella, Inc. 4595 Mountain Lakes Boulevard Redding CA 96003 USA Phone: + 530 243 2200 Fax: + 530 243 3761 E-mail: sales@tedpella.com Web: www.tedpella.com
Ordering Distribution
info@buybbi.com â&#x20AC;˘ Buy products online at www.buybbi.com
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