Western Blotting Some protein detection kits make you wonder if your eyes are playing tricks on you.
With KPL’s Protein Detector™ Western Blotting Systems, the results jump right out at you.
Western Blotting No matter which KPL Western Blotting system you use, you’ll get a strong, clean signal every time. KPL’s blotting substrates and Protein Detector™ Western Blot Kits provide consistent, high quality results when analyzing proteins immobilized on membrane. Choose from a line of substrates and kits for either chemiluminescent or colorimetric methods of detecting specific antigens in a complex protein mixture. The cornerstone of KPL’s Western blotting systems is our line of sensitive liquid substrates. You have a choice among multiple chemiluminescent and chromogenic substrates
for peroxidase and phosphatase that provide varied levels of sensitivity. The chemiluminescent substrates provide a reliable alternative to conventional colorimetric ELISA with the advantage of increased sensitivity. Our chemiluminescent substrates for alkaline phosphatase detection, PhosphaGLO™ and PhosphaGLO Reserve™ AP Chemiluminescent Substrates, offer sensitivity and duration of signal in a one-component system.
KPL’s substrates are offered as standalone reagents or combined with unique blocking and wash solutions and highly specific secondary antibodies to create comprehensive, fully optimized kits for Western blotting. Each of these kits is designed to deliver the maximum signal-to-noise ratio available. Choose the detection system that best meets your needs and achieve clean, clear results blot after blot.
Substrates for Western Blotting KPL’s Western blotting substrates for use in peroxidase and phosphatase reporter systems offer high quality, reproducible chemiluminescent and colorimetric detection. It’s your choice! LumiGLO Reserve™ HRP Substrate
LumiGLO® HRP Substrate
PhosphaGLO Reserve™ AP Substrate
PhosphaGLO™ TMB HRP AP Substrate Substrate
4 CN Substrate
BCIP/NBT AP Substrate
FirePhos™ Membrane AP Substrate
Type
chemi.
chemi.
chemi.
chemi.
color.
color.
color.
color.
Format
2-comp
2-comp
1-comp
1-comp
1- or 3comp
2-comp
1- or 3comp
1-comp
Detection Method
film or image analysis
film or image analysis
film or image analysis
film or image analysis
visual-dark blue visual - purple precipitate precipitate
visual - purple precipitate
visual - red
Detection Limit
Sub-picogram; femtogram
0.6 picogram
femtogram
picogram
50 picogram
500 picogram
low nanogram
low nanogram
Stability of Working Solution
8 hours
24 hours
2 years at 4o C
2 years at 4o C
1-comp1 year at 4o C. 3-comp24 hours
1 hour
1-comp1 year at 4o C. 3-comp1 hour
NA
Duration of Signal
4 - 8 hours
1 - 2 hours
5 days
5 days
NA
NA
NA
NA
Enzyme Catalyst
HRP
HRP
AP
AP
HRP
HRP
AP
AP
Kinetics
5 minutes
5 minutes
15 minutes
15 minutes
10 minutes
10 minutes
10 minutes
10 minutes
Recommended Membrane
nitrocellulose or PVDF
nitrocellulose or PVDF
nitrocellulose or PVDF
nitrocellulose or PVDF
nitrocellulose or PVDF
nitrocellulose or PVDF
nitrocellulose or PVDF
nitrocellulose or PVDF
comp = component • chemi. = chemiluminescent • color. = colorimetric • HRP = peroxidase • AP = phosphatase • NA = not applicable
www.kpl.com
Western Blotting Chemiluminescent Substrates and Western Blot Kits Rapid, Sensitive Detection with Luminol-based Substrates Chemiluminescent detection involves the creation of light through the catalysis of an enzyme substrate. Use of this method for protein detection allows an increase in sensitivity by orders of magnitude compared to traditional colorimetric Western or dot blotting. KPL offers two unique luminol-based chemiluminescent substrates —LumiGLO® and LumiGLO Reserve™, for the rapid and sensitive detection of horseradish peroxidase (HRP)-labeled conjugates. Maximum Sensitivity with LumiGLO ReserveTM LumiGLO Reserve HRP Chemiluminescent Substrate offers an option for those assays where enhanced sensitivity is critical to success. This proprietary two-component substrate formulation provides greater than 20 times the sensitivity of standard LumiGLO with detection levels as low as the femtogram range (Figure 1).
Long Signal Duration LumiGLO Reserve emits light over the course of 4-8 hours with the most intense emission occurring within the first two hours. Because of its extreme light intensity, most images may be captured well under 10 minutes; multiple exposures are easy to obtain. High signal intensity facilitates the detection of
Convenience LumiGLO Reserve Chemiluminescent Substrate Kits are supplied in several kit formats to meet diverse user needs. This substrate can replace your current chemiluminescent substrate in existing assays where greater sensitivity is desired. LumiGLO Reserve Western Blot Kit LumiGLO Reserve is also offered as part of a fully optimized kit, the Protein Detector LumiGLO Reserve Western Blot Kit, providing femtogram detection of proteins immobilized on membranes. For assurance against background interference, this kit contains our unique Detector™ Block for optimal signal-to-
both high and low abundance proteins and makes it an ideal system for use with chemiluminescent imagers. Sample and Antibody Conservation LumiGLO Reserve provides the added benefit of strong signal with the use of reduced amounts of precious target and antibodies. Therefore, material of limited supply or higher expense can be conserved while maintaining your current level of sensitivity.
noise ratio; very low background can be achieved without compromise to signal intensity (Figures 2 and 3). Detector Block is also offered separately.
Superior Signal to Noise LumiGLO Reserve delivers lower nonspecific signal than competitor substrates in its class (Figures 2 and 3).
Reliable and Economical LumiGLO® KPL’s original LumiGLO Chemiluminescent Substrate provides high quality results in a variety of immuoassays.
LumiGLO Reserve Signal-to-Noise Comparison 1
2
3
4
5
6
A
LumiGLO B
LumiGLO Reserve C
ECL Plus
www.kpl.com
Figure 1: Relative expression of transcription factor, c-myc, using different chemiluminescent substrates. Five two-fold serial dilutions of purified c-myc (25 ng–1.56 ng, lanes 1–5) were compared to a 64 µg total protein HeLa nuclear lysate (lane 6). Following separation on a 4-20% PAGE gel and transfer to PVDF, protein was detected using a rabbit anti-c-myc antibody (1:200) and anti-rabbit HRP conjugate (1:10,000). Detection conditions were identical with the exception of substrate. While the c-myc lysate sample was not detectable with A) LumiGLO or C) ECL Plus™ after 10 minutes, the sample was easily detected with B) LumiGLO Reserve after just a 2-minute film exposure.
A
LumiGLO Reserve
B
ECL Plus
C
ECL Advance
Figure 2: Comparison of low-end sensitivity using LumiGLO Reserve and ECL Detection Kits. Two-fold serial dilutions of Mouse IgG (1 ng – 31 pg) were separated by SDS-PAGE and transferred to PVDF. Under manufacturer’s recommended conditions, protein was detected using HRP-labeled anti-mouse antibody (varied dilutions according to recommended optimization) and each respective substrate: A) LumiGLO Reserve, B) ECL Plus™, C) ECL Advance™. Film was exposed for 10 minutes and analyzed for sensitivity and signal to noise.
LumiGLO vs. Leading Traditional Chemiluminescent Substrates Densitometry for LumiGLO vs. ECL
Superior Signal to Noise with Detector™ Block
50000 LumiGLO
45000
ECL
Relative Image Density
40000 35000 30000 25000
LumiGLO Chemiluminescent Peroxidase Substrate
Supersignal® West Pico
ECL Plus™
ECL™
20000 15000 10000 5000 0 0
50
100
150
200
250
Concentration (ng)
Figure 4: Signal response comparison of LumiGLO vs. ECL™ chemiluminescent substrates in Western blotting. ß-galactosidase was electrophoresed, transferred to PVDF membrane and subsequently detected using rabbit anti-ß-gal followed by HRP conjugated goat anti-rabbit IgG (H+L). Each blot was treated with 5 mL of LumiGLO or ECL substrate and exposed to film for 10 minutes. The density of each band was analyzed on a Syngene GeneGenius™ image analyzer, using automatic background subtract.
Researchers continue to select LumiGLO for reliable performance in Western blot detection. Ideal for Routine Western Blotting
LumiGLO detects low picogram quantities of target protein on blots. After reaction with membrane-bound HRP conjugates, light emission begins immediately and continues for approximately 1–2 hours. Detection is possible within minutes of blot exposure.
Figure 5: Detection of ß-galactosidase in Western blots using alternative chemiluminescent substrates. A two-fold dilution series of purified ß-galactosidase (from 25 was electrophoresed on a polyacrylamide gel and transferred to KODAK BIOMAX Multi-Blot Kit for Proteins. Each blot was detected under the same conditions using Protein Detector LumiGLO Western Blotting Kit, substituting 5 mL LumiGLO on 3 of the 4 blots with 5 mL of leading competitive chemiluminescent substrates, respectively. Following a 10 minute film exposure, results were evaluated for sensitivity and signal:noise ratio.
Greater Linearity
LumiGLO produces a quantitatively linear signal on film that ensures a broader dynamic range of detection. While the light output from other chemiluminescent substrates tends to reduce sharply as the concentration of protein is titrated, a proportional reduction in sensitivity is achieved with LumiGLO (Figure 4). Low Background
LumiGLO delivers superior signal-tonoise by design. When used with KPL’s Detector™ Block, nonspecific binding is
further reduced and ensures low background without loss of signal intensity. Figure 5 compares the superior signalto-noise from blots blocked with Detector Block and subsequently detected with LumiGLO to leading competitor substrates. Cost Effective
LumiGLO is more economical blot for blot compared to competitive products. Enjoy the benefits of a sensitive, reliable substrate while staying within budget. LumiGLO Western Blot Kit
Figure 3: Signal linearity and signal-to-noise ratio comparison of advanced chemiluminescent substrates. Mouse IgG (250 pg to 3.91 pg) was transferred to nitrocellulose. Blots were detected under identical conditions with a 1:10,000 dilution of HRP-Goat-anti-Mouse IgG (except ECL Advance, 1:100,000) and exposed to film for 10 minutes. Densitometry was then performed using the Syngene GeneGenius. LumiGLO Reserve demonstrates superior signal linearity over a larger dynamic range and the greatest comparative signal-to-noise.
The LumiGLO Western Blot Kit is designed for low picogram detection of proteins immobilized on membranes. Researchers choose this kit for its reliable, consistent performance. The combination of a stable, liquid conjugate and a sensitive chemiluminescent substrate allows rapid and accurate identification of samples. For assurance against background interference, this kit contains our unique Detector Block for optimal signal-to- noise ratio (Figure 5).
Power Your Immunoassay
Western Blotting Sensitivity and Convenience with PhosphaGLOTM AP Substrates KPL offers two chemiluminescent substrates for use with alkaline phosphatase, PhosphaGLO ReserveTM and PhosphaGLOTM AP Substrates. PhosphaGLO Reserve Substrate overcomes the limitations posed by conventional chemiluminescent substrates for AP. With sensitivity in the femtogram range, PhosphaGLO Reserve enables detection of target protein in low concentrations. PhosphaGLO AP Substrate is recommended for routine detection of proteins in the picogram range (Figure 6). Low Background without Special Blockers
PhosphaGLO Substrates produce superior signal and low background, providing a better signal to noise ratio and a cleaner image than other chemiluminescent substrates for AP (Figure 6). No special blockers are needed with either nitrocellulose or PVDF membrane. Ultimate Convenience
Both PhosphaGLO AP Substrates offer exceptional convenience as one-component solutions ready to use. They are stable for up to two years when stored at 4o C. Their long glow times of 5 days facilitate assay development, enabling repeat exposures.
PhosphaGLO Reserve AP Substrate
PhosphaGLO AP Substrate
Colorimetric Kits
Western Blot Reagents
KPL also offers colorimetric Western blot systems when chemiluminescent detection is not preferred. Results can be interpreted by direct visualization of the blot. Everything you need to detect your antigen and primary antibody is supplied for convenience and optimal performance.
In addition to our line of substrates and kits, KPL offers a broad line of secondary antibodies, conjugates and support reagents in various packaging formats to suit your needs.
Protein DetectorTM TMB Western Blot Kit The Protein DetectorTM TMB Western Blot Kit utilizes TMB Peroxidase Membrane Substrate (3,3’,5,5’-tetramethylbenzidine), the most sensitive chromogenic peroxidase substrate for Western and dot blotting applications. Detection limits are significantly increased as compared to other chromogenic membrane substrates. TMB produces a dark blue precipitate upon reaction with HRP.
Secondary Antibodies
• Highly purified and specific. • Large offering of species-specific polyclonal antibodies • Enzyme, biotin and fluorochrome-labeled antibodies Support Reagents
• One source for block solutions, wash buffers, diluents and stabilizers. • Consistently produced for reliable results.
Protein DetectorTM BCIP/NBT Western Blot Kit For detection of phosphatase-labeled conjugates, the Protein Detector BCIP/NBT Western Blot Kit is ideal. When reacted with alkaline phosphatase, BCIP/NBT produces clean, intense bands of purple precipitate. It also provides stable, more permanent results than other chromogenic substrates.
CDP Star® Chemiluminescent Substrate
Figure 6: Comparison of low-end sensitivity using PhosphaGLO Reserve AP Substrate, PhosphaGLO AP Substrate, and CDP-Star Chemiluminescent Substrate on PVDF membranes. Five-fold serial dilutions of mouse IgG (2 ng -3.2 pg) were separated by SDS-PAGE and transferred to membranes. Protein was detected using a 1:1000 dilution of biotin-labeled goat anti-mouse IgG, a 1:10,000 dilution of AP-labeled streptavidin and each respective substrate. Film was exposed for 10 minutes.
LumiGLO is a registered trademark and LumiGLO Reserve, Protein Detector, PhosphaGLO, PhosphaGLO Reserve, FirePhos and Detector are trademarks of KPL, Inc. ECL, ECL Plus and ECL Advance are trademarks of GE Healthcare. SuperSignal West is a registered trademark of Pierce Biotechnology, Inc. BIOMAX is a trademark of KODAK. GeneGenius is a trademark of Syngene. Biodyne is a registered trademark of Pall/Gelman Corporation. CDP-Star is a registered trademark of Applied Biosystems.
Power Your Immunoassay
DyLightTM Fluorescent Conjugates KPL’s DyLightTM Conjugates - A Brilliant Choice! KPL’s DyLightTM conjugates offer a brilliant choice in a variety of multicolor detection applications, including fluorescence microscopy, flow cytometry, Western blotting, ELISA and array platforms. Our affinity purified antibodies combined with a series of outstanding DyLight dyes provide superior performance over conventional CyDyeTM fluors, fluorescein and rhodamine, with performance comparable to that of Alexa Fluor® dyes (Figure 1). Enjoy these advantages when you switch to KPL’s DyLight Conjugates:
KPL offers eight DyLight dyes, including 405, 488, 549, 594, 633, 649, 680 and 800 with well-differentiated excitation and emission spectra. Our extensive line of over 170 DyLight conjugates is available across a range of animal species immunoglobulin, including human, mouse, rabbit, rat, other species and streptavidin. See back cover to find out what sets KPL’s antibodies apart. Comparison of DyLight 649 and Alexa 647 Conjugates in a Performance Assay
• High sensitivity, bright signal enable conjugate • • • • • •
conservation More photostable than Cy, FITC; comparable to Alexa High signal-to-noise ratios; low background Wide selection of specific antibodies Consistent, lot-to-lot performance Narrow emission spectra enable specific, multicolor analysis Compatible with a variety of buffers.
Antigen Concentration (ng/mL)
Figure 1. KPL DyLight 649 conjugates demonstrate comparable fluorescense intensity and photostability to Alexa 647 conjugates in a FLISA.
Image above: HMVEC-L primary endothelial cells. F-actin detected with DY554-Phalloidin (rendered green). Microtubules detected with anti-tubulin antibody and DyLight 649 conjugated goat anti-mouse IgG (red). Nuclei detected with DAPI (blue)
Power Your Immunoassays
DyLightTM Fluorescent Conjugates DyLight Conjugated Affinity Purified Antibodies and Streptavidin KPL DyLight conjugates provide a choice of outstanding fluorescent conjugates with absorption spectra ranging from 405 nm to 800 nm. Their emission profiles correspond to those of other commonly used fluorophores such as Alexa, FITC and the Cy dyes. Narrow, defined emission spectra enable the use of multiple fluorescence labeling and simultaneous identification of several target molecules in the same sample. Light output is comparable to IRDye and Alexa and more intense than Cy Dyes, FITC or TRITC. DyLight dyes demonstrate resistance to photobleaching, resulting in excellent photostability as well as high solubility in aqueous solutions across a range of pH values.
Emission
DyLight Dye
Ex/Em(nm) Replaces
Blue
DyLight 405
400/420
Alexa 405 and Cascade Blue
Green
DyLight 488
493/518
Alexa 488 and FITC
Yellow
DyLight 549
550/568
Alexa 546, Alexa 555, Cy3, TRITC
Orange
DyLight 594
593/618
Alexa 594, Texas Red
Red
DyLight 633
638/658
Alexa 633
Red
DyLight 649
646/674
Alexa 647, Cy5
Near IR
DyLight 680
682/715
Alexa 680, Cy5.5
Infrared
DyLight 800
770/794
IRDye 800
nm = nanometers IR = infrared
Ordering Information Description Anti-Mouse IgG (γ), HSA Anti-Mouse IgG (H+L), HSA Anti-Mouse IgG (H+L), HSA, 0.1 mg Anti-Mouse IgG (H+L), RbSA, HSA Anti-Mouse IgM (μ), HSA Anti-Mouse IgG+IgM (H+L), HSA F(ab’)2 Anti-Mouse IgG (γ), HSA F(ab’)2 Anti-Mouse IgG (H+L), HSA Anti-Human IgG (H+L) Anti-Human IgG (H+L), 0.1 mg Anti-Human IgG (γ) Anti-Human IgM (μ) F(ab’)2 Anti-Human IgG (γ) F(ab’)2 Anti-Human IgM (μ) F(ab’)2 Anti-Human IgG (H+L) Anti-Rabbit IgG (H+L) Anti-Rabbit IgG (H+L), 0.1 mg Anti-Rabbit IgG (H+L), HSA F(ab’)2 Anti-Rabbit IgG (H+L), HSA Anti-Rat IgG (H+L) Anti-Rat IgG (H+L), 0.1 mg Anti-Guinea Pig IgG (H+L) Anti-Chicken IgG (H+L) Anti-Dog IgG (H+L) Anti-Goat IgG (H+L) Anti-Horse IgG (H+L) Rabbit Anti-Sheep IgG (H+L) Anti-Swine IgG (H+L) Streptavidin Streptavidin, 0.1 mg
Power Your Immunoassay
DyLight 405
072-08-18-06 042-08-18-06
202-08-18-06 072-08-10-06 042-08-10-06
202-08-10-06 072-08-15-06 042-08-15-06 202-08-15-16 072-08-16-06
072-08-24-06 072-08-13-06
072-08-30-00 042-08-30-00
DyLight 488
DyLight 549
072-03-18-02 072-03-18-06 042-03-18-06 072-03-18-18 072-03-18-03 072-03-18-09 202-03-18-02 202-03-18-06 072-03-10-06 042-03-10-06 072-03-10-02 072-03-10-03 202-03-10-02 202-03-10-03 202-03-10-06 072-03-15-06 042-03-15-06 072-03-15-16 202-03-15-16 072-03-16-06 042-03-16-06 072-03-17-06 072-03-24-06 072-03-19-06 072-03-13-06 072-03-21-06 072-03-23-06 072-03-14-06 072-03-30-00 042-03-30-00
072-04-18-02 072-04-18-06 042-04-18-06 072-04-18-18 072-04-18-03 072-04-18-09 202-04-18-02 202-04-18-06 072-04-10-06 042-04-10-06 072-04-10-02 072-04-10-03 202-04-10-02 202-04-10-03 202-04-10-06 072-04-15-06 042-04-15-06 072-04-15-16 202-04-15-16 072-04-16-06 072-04-17-06 072-04-24-06 072-04-19-06 072-04-13-06 072-04-21-06 072-04-23-06 072-04-14-06 072-04-30-00 042-04-30-00
DyLight 594
DyLight 633
072-09-18-06 042-09-18-06
072-10-18-06 042-10-18-06
202-09-18-06 072-09-10-06 042-09-10-06
202-10-18-06 072-10-10-06 042-10-10-06
202-09-10-06 072-09-15-06 042-09-15-06
202-10-10-06 072-10-15-06 042-10-15-06
202-09-15-16 072-09-16-06
202-10-15-16 072-10-16-06
072-09-24-06
072-10-24-06
072-09-13-06
072-10-13-06
072-09-30-00 042-09-30-00
072-10-30-00 042-10-30-00
DyLight 649 072-05-18-02 072-05-18-06 042-05-18-06 072-05-18-18 072-05-18-03 072-05-18-09 202-05-18-02 202-05-18-06 072-05-10-06 042-05-10-06 072-05-10-02 072-05-10-03 202-05-10-02 202-05-10-03 202-05-10-06 072-05-15-06 042-05-15-06 072-05-15-16 202-05-15-16 072-05-16-06 072-05-17-06 072-05-24-06 072-05-19-06 072-05-13-06 072-05-21-06 072-05-23-06 072-05-14-06 072-05-30-00 042-05-30-00
DyLight 680
072-06-18-06 042-06-18-06 072-06-18-18
202-06-18-06 072-06-10-06 042-06-10-06
202-06-10-06 072-06-15-06 042-06-15-06 072-06-15-16 202-06-15-16 072-06-16-06 072-06-17-06 072-06-24-06 072-06-19-06 072-06-13-06 072-06-21-06 072-06-23-06 072-06-14-06 072-06-30-00 042-06-30-00
Near Infrared Fluorophores DyLight 680 and 800 Conjugates Offer Sensitive Multicolor Imaging in Western Blotting
DyLight Dyes Emission Spectra
DyLight 680 and 800 dyes emit in the near infrared and infrared ranges of the light spectrum respectively and are ideal for multicolor protein detection in Western blotting. Unlike fluorescent conjugates that emit in the visible range, DyLight 680 and 800 conjugates provide a unique set of advantages: • Secondary antibodies with defined specificity and sensitivity • Brighter signal than visible fluorescence • Virtually no background autofluorescence from membranes or most biological specimens Effective Alternative to Chemiluminescence • Broader dynamic range than chemiluminescence • Quantitation accuracy superior to traditional methods • Easy to assay multiple proteins simultaneously on one Western blot • Cost-effective - eliminates need for chemiluminescence substrates, film and darkroom • Clean results - no bleeding from consecutive lanes
ht 800
07-18-06 07-18-06 07-18-18
Near Infrared Fluorescent Imaging with Secondary Antibodies labeled with DyLight 680 and 800
07-18-06 07-10-06 07-10-06
07-10-06 07-15-06 07-15-06 07-15-16 07-15-16 07-16-06 07-17-06 07-24-06 07-19-06 07-13-06 07-21-06 07-23-06 07-14-06 07-30-00 07-30-00
Figure 2. DyLight 680 anti-mouse IgG and DyLight 800 anti-rabbit IgG secondary antibody conjugates provide low background and high signal in two-color Western blot detection of tubulin and TNFα. Membrane was imaged with the LI-COR Odyssey Infrared Imaging System. HSA = human serum adsorbed RbSA = rabbit serum adsorbed DyLight antibody conjugates are made in goat except anti-goat and anti-sheep antibodies are made in rabbit. Supplied in 1.0 mg lyophilized form except select 0.1 mg sizes.
1.800.638.3167
www.kpl.com
Immunofluorescence using DyLight Conjugates See bright fluorescence and low background with KPL DyLight conjugates in immunohistology applications. The winning combination of DyLight dyes and KPL purified antibodies enables multicolor labeling of two or more targets with similar intensity and photostability to Alexa dyes without the limitations of fluorescein and CyDyes.
Mouse primary cortical neurons MCx WCS stained with DyLight 549 (red). Synaptophysin stained with DyLight 488 (green).
HMVEC-L primary endothelial cells F-actin detected with DY554-Phalloidin (rendered green). Microtubules stained with DyLight 649 (red). Nuclei detected with DAPI (blue).
Rat hippocampal neurons WCS stained with; DyLight 488 (green). MAP2 stained with DyLight 549 (red).
A549 cells Cytokeratin stained with DyLight 680 (pseudocolored white). Lamin A stained with DyLight 549 (pseudocolored red).
NIH 3T3 cells F-actin detected with DY554-Phalloidin (rendered green). Microtubules stained with DyLight 649 (red). Nuclei detected with DAPI (blue).
Cells were probed with anti-lamin-A and rabbit anti-cytokeratin 18. Detected with a mixture of DyLight 405labeled goat anti-mouse and DyLight 633-labeled goat anti-rabbit antibodies.
Cells were probed with anti-lamin-A and rabbit anti-cytokeratin18. Detected with a mixture of DyLight 488-labeled goat anti-mouse and DyLight 405labeled goat anti-rabbit antibodies.
Codetection of mouse anti-α-tubulin and rabbit anti-lamin B1 using DyLight 594-labeled goat antimouse and DyLight 488-labeled goat anti-rabbit antibodies.
To order or for more information on KPL’s line of unlabeled and conjugated
DyLightTM Fluorescent Conjugates KPL AntibodiesWhat Sets Them Apart In 1979, KPL pioneered the production of large-scale affinity purification and was the first company to commercialize affinity purified secondary antibodies. Rigorous standards throughout the antibody production process make our antibodies standout in the marketplace. Many manufacturers cut corners by beginning with inferior serum and extracting the useful antibody towards the end of their process. KPL spends considerable effort developing and purifying its own immunogen formulation to generate the antiserum, because pure immunogen results in a more potent and specific antibody prior to any purification steps. Further, ISO 9001:2008-certified quality procedures are carried out at more than six different stages of the antibody production cycle, and material that does not meet our high standards for potency and cross-reactivity is rejected. Our late stage purification process has been continually refined since 1979 and relies on a one of a kind custom column resin which is uniquely suited to our antibody manufacturing process. Finally, our process of pooling antiserum from multiple animals tempers natural serum variability, minimizing variances from animal-to-animal. The result is more standardized large-scale antibody lots with increased consistency. KPL’s experience, innovative processes, and attention to detail result in highperformance antibodies that are unique.
Gaithersburg, MD Phone: 800.638.3167/301.948.7755 Fax: 301.948.0169 www.kpl.com
ISO 9001:2008 Registered
affinity purified antibodies, contact us at 800.638.3167/301.948.7755, fax 301.948.0169 or visit www.kpl.com. ML356-04 For research use only. DyLight is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries. Cy and Cy Dye are trademarks of GE Healthcare. Alexa Fluor is a registered trademark of Invitrogen. ©2009 KPL, Inc. All rights reserved.
Power Your Immunoassays
Using LumiGLO
Using LumiGLO Reserve™
LumiGLO Reserve™ LumiGLO Reserve™ Chemiluminescent Substrate and Western Blot Kits See More of What You’ve Been Missing! For years, researchers have relied on LumiGLO® Chemiluminescent Substrate for picogram detection of protein on Western blots. KPL has expanded this line to now include a new class of HRP-based chemiluminescent kits – LumiGLO Reserve – for maximum sensitivity in Western blot detection. Take a closer look at the advantages of LumiGLO Reserve: Maximum Sensitivity
LumiGLO Reserve’s proprietary two-component substrate formulation allows for greater than 20 times the sensitivity than standard LumiGLO. With detection levels in the femtogram range, LumiGLO Reserve is ideal for those situations where the protein of interest is expressed in low abundance. Sample and Antibody Conservation
LumiGLO Reserve provides the added benefit of strong signal with the use of reduced amounts of precious target and antibodies. Therefore, material of limited supply or higher expense can be conserved while maintaining your current level of sensitivity.
Superior Signal to Noise
In combination with KPL’s proprietary Detector™ Block, very low background can be achieved without compromise to signal intensity. Convenience
LumiGLO Reserve is supplied in several kit formats to offer the most flexibility. A comprehensive Protein Detector LumiGLO Reserve Western Blot Kit is available for a fully optimized approach to chemiluminescent Western blot detection. Alternatively, LumiGLO Reserve Chemiluminescent Substrate Kits can be used in existing assays where greater sensitivity is desired. 1
2
3
4
5
6
A
B
C
Figure 1: Relative expression of transcription factor, c-myc, using different chemiluminescent substrates. Five two-fold serial dilutions of purified c-myc (25 ng – 1.56 ng, lanes 1–5) were compared to a 64 µg total protein HeLa nuclear lysate (lane 6). Following separation on a 4-20% PAGE gel and transfer to PVDF, protein was detected using a rabbit anti-c-myc antibody (1:200) and anti-rabbit HRP conjugate (1:10,000). Detection conditions were identical with the exception of substrate. While the c-myc lysate sample was not detectable with A) LumiGLO or C) ECL Plus after 10 minutes, the sample was easily detected with B)LumiGLO Reserve after just a 2-minute film exposure.
Power Your Immunoassays
LumiGLO Reserve™ LumiGLO® Chemiluminescent Substrate Selection Guide LumiGLO Reserve
LumiGLO
Detection Limit
Femtogram
Picogram
Emission Duration
4–8 hours
1–2 hours
Stability of Working Solution
8 hours at RT
24 hours at RT
Recommended Membrane
Nitrocellulose or PVDF
Nitrocellulose or PVDF
Recommended Detection Method
Film/Chemiluminescent Imager
Film
Technical Tips
Comparison of HRP Chemiluminescent Detection Reagents
LumiGLO Reserve™ was compared to leading high sensitivity HRP chemiluminescent substrates to determine the relative signal provided when detecting protein of reduced concentrations. Each detection reagent was used according to the manufacturers’ recommended protocols as well as under identical conditions. A
B
In either case, LumiGLO Reserve was able to detect significantly lower protein concentrations than most competitive substrates. In addition, the LumiGLO Reserve kits delivered the best signal to noise compared to the other systems tested (See Figures 2 and 3).
B
• LumiGLO Reserve emits light over the course of 4–8 hours with the most intense emission within the first two hours. Because of its high light intensity, most images may be captured well within 10 minutes making multiple exposures easy to obtain.
C
• The increased light output also makes this substrate ideal for use with chemiluminescent imagers. LumiGLO Reserve has been tested on Alpha Innotech FluorChem™ 8000 and Syngene GeneGnome™ instruments.
Figure 3: Comparison of low-end sensitivity using LumiGLO Reserve and ECL Substrates. Western blots of the same serial dilutions of Mouse IgG were prepared as in Figure 2. Under identical block and wash conditions, protein was detected using HRP-labeled anti-mouse antibody (1:10,000 of 0.1 mg/mL stock) and each respective substrate: A) LumiGLO Reserve, B) ECL Plus, C) ECL Advance. Film was exposed for 30 seconds and analyzed for sensitivity and signal to noise.
Ordering Information Catalog No. 54-71-00
• Because of LumiGLO Reserve’s sensitivity, it is imperative to the success of the assay that the primary antibody and HRP conjugate be titrated to give the optimal signal to noise. In many cases, antibodies can be used at more dilute concentrations than previously used with other chemiluminescent substrates.
C
Figure 2: Comparison of low-end sensitivity using LumiGLO Reserve and ECL Detection Kits. Two-fold serial dilutions of Mouse IgG (1 ng – 31 pg) were separated by SDS-PAGE and transferred to PVDF. Under identical conditions, protein was detected using HRP-labeled anti-mouse antibody (varied dilutions according to recommended optimization) and each respective substrate: A) LumiGLO Reserve, B) ECL Plus™, C) ECL Advance™. Film was exposed for 10 minutes and analyzed for sensitivity and signal to noise.
A
• LumiGLO Reserve can be used with nitrocellulose and PVDF membranes.
Product Name LumiGLO Reserve Chemiluminescent Substrate Kit
Size 2400 cm2
Kit Contents: LumiGLO Reserve Substrate Solutions A and B, and Wash Solution Concentrate.
54-71-01
LumiGLO Reserve Chemiluminescent Substrate Kit
600 cm2
Kit Contents: LumiGLO Reserve Substrate Solutions A and B, and Wash Solution Concentrate. Gaithersburg, MD 20878
54-13-50
Protein Detector™ LumiGLO Reserve Western Blot Kit
2400 cm2
Kit Contents: LumiGLO Reserve Substrate Solutions A and B, Wash Solution Concentrate, Detector Block, and HRP-labeled Anti-Rabbit and Anti-Mouse Conjugates.
To order or for more information on KPL’s full line of protein and nucleic acid detection products, contact us at 800.638.3167 / 301.948.7755, FAX 301.948.0169 or visit us at www.kpl.com.
Phone: 800.638.3167 Fax: 301.948.0169 www.kpl.com
ISO 9001:2008 Registered
LumiGLO is a registered trademark and Detector, Protein Detector and LumiGLO Reserve are trademarks of KPL, Inc. ECL Plus and ECL Advance are trademarks of Amersham Biosciences UK Limited. FluorChem is a trademark of Alpha Innotech Corporation. GeneGnome is a trademark of Syngene. © 2003 KPL, Inc. All rights reserved. ML279-05
Power Your Immunoassays
HRP Membrane Color Substrates Sensitivity, reliability and affordability! KPL offers two substrates commonly used to detect
price. The substrate may be adapted as a soluble sub-
horseradish peroxidase (HRP) conjugated antibodies
strate for ELISA by omitting TMB Membrane
that bring complementary benefits to membrane
Enhancer. Both substrates perform equally well on
detection; TMB (3, 3´, 5, 5´-tetramethylbenzidene)
nitrocellulose and PVDF membranes.
Membrane Substrate and 4 CN (4-chloro-1-naphthol) Membrane Substrate. TMB substrate offers the most
4 CN produces a distinct, blue-purple precipitate and extremely low background.
sensitive colorimetric detection for blotting applications and is recommended when high signal and low background are required. 4 CN substrate is highly reliable and is notable for its characteristically low
TMB produces a blue precipitate and provides greater sensitivity than 4 CN.
background. •
TMB Membrane Substrates (1- and (3-Component) 4 CN
TMB
In the presence of peroxidase conjugate, KPL’s TMB Membrane Substrates produce a stable, dark blue pre-
• 4 CN Peroxidase Substrate (2-Component)
cipitate at the site of a positive reaction, enabling detection of as little as 50 pg of peroxidase. Our TMB
4 CN (4-chloro-1-napthol) Peroxidase Membrane
substrates are available as 1- and 3- component solu-
Substrate produces a purple precipitate at the reaction
tions. 1-component TMB Peroxidase Substrate is ready
site in the presence of peroxidase conjugates. Due to
to use and contains hydrogen peroxide and buffer.
its low background, 4 CN presents a desirable alter-
Our 3-component TMB contains TMB in an organic
native to other, more sensitive substrates when back-
base, H2O2 in a citric acid buffer and a third component, TMB Membrane Enhancer. It provides sensitivi-
ground poses a problem.
ty equivalent to 1-component TMB at an economic
Power Your Immunoassays
Support Reagents Benefits of KPL’s HRP Membrane Substrates •
KPL provides a broad range of assay support reagents for Western blot detection. Conjugate stabilizers, diluents, block and wash solutions are offered in convenient stable liquid form to ensure accurate reproducible results, blot after blot. Our Detector Block is ideal for use as a diluent/block solution for Western blots, Southern blots and Northern blots on a variety of membranes.
TMB Peroxidase Membrane Substrate
• • • • • • • •
Most sensitive colorimetric substrate for blotting Delivers results at the picogram level Recommended for both kinetic and endpoint ELISA Contains no harmful organic solvents; safer and more sensitive than DAB or AEC. 1-Component TMB-no preparation required. Ensures more consistent results 3-Component TMB-same performance as 1-Component TMB at an economic price. Blots remain stable with minimal fading
4 CN Peroxidase Substrate
• • •
• DetectorTM Block
Easy mix-and-use, two-component solutions Very low background, moderate sensitivity High contrast image ideal for photography and publication
Ordering Information Catalog#
Description
Size
50-77-18
TMB Membrane Substrate (1-Component)
100 mL
50-77-03
TMB Membrane Substrate (1-Component)
200 mL
50-77-04
TMB Membrane Substrate (1-Component)
1L
50-77-00
TMB Membrane Substrate (3-Component)
440 mL
50-77-01
TMB Membrane Enhancer
50-73-00
4 CN Substrate System (2-Component)
600 mL
50-73-04
4 CN Substrate System (2-Component)
2700 mL
Detector Block is recommended as a general blocking agent in membrane-based detection assays. It is especially useful when traditional blocking solutions (i.e., milk, BSA, casein) reduce sensitivity or do not adequately block background. Detector Block can be used as both a blocking solution and a conjugate diluent in membrane applications.
40 mL
Protein Detector TMB Western Blot Kit 54-11-50
Protein Detector TMB Western Blot Kit
2500 cm2
Detector Block 71-83-00
Detector Block
240 mL
Gaithersburg, MD 20878 Phone: 800.638.3167 Fax: 301.948.0169
To order or for more information on KPL’s line of protein detection products, contact us at 800.638.3167 / 301.948.7755, fax 301.948.0169 or visit us at www.kpl.com.
www.kpl.com
ISO 9001:2008 Registered
Protein Detector is a trademark of KPL. ©2008 KPL, Inc. All rights reserved. ML353-02 Power Your Immunoassays
Peroxidase Conjugates KPL Peroxidase Conjugates: Time-tested, Sensitive and Reliable Since 1979 KPL has provided quality affinity
streptavidin. They are affinity purified and in
purified antibodies to researchers worldwide.
some cases further adsorbed to minimize cross-
Over the years we have refined our production
reactivity between animal species or shared
process to provide antibodies with high potency
reactivity with other immunoglobulin classes.
and consistent performance in immunoassays.
HRP-labeled F(ab’)2 fragment antibodies are
From the start KPL gives careful consideration
offered for assays requiring extremely low back-
to immunogen preparation, using a highly
ground and absence of F(c)-mediated binding.
purifed formulation to generate antiserum. KPL pools antiserum from multiple animals to
KPL reacts HRP of the highest quality with
reduce natural animal to animal serum variabili-
affinity purified antibodies and streptavidin
ty. During the purification process our ISO
using the periodate method of Nakane and
9001:2008-certified quality procedures impose
Kawaoi. Special features of HRP include:
rigorous standards for potency and cross-reac-
• faster catalytic rate than alkaline phosphatase • generates more product in shorter incubation
tivity. The result is standardized antibody lots with excellent reproducibility. Our extensive line of peroxidase (HRP) conjugates is available across a range of animal species, including human, mouse, rabbit and rat antibodies, as well as other animal species and
times
• provides maximum sensitivity, low nonspecific binding
• ideal for ELISA, Western blotting and immunohistology applications. Power Your Immunoassays
Peroxidase Conjugates Peroxidase Conjugates Ordering Information (Partial listing) Catalog#
Description
Size
KPL offers a range of sensitive substrates for use with HRP conjugates. They provide a choice of intense colors for ELISA, blotting and cell staining applications.
04-10-06
HRP-labeled Goat Anti-Human IgG (H+L)
0.1 mg
04-10-17
HRP-labeled Goat Anti-Human IgA+IgG+IgM (H+L), MSA
0.1 mg
04-10-20
HRP-labeled Goat Anti-Human IgG (Fc)
0.1 mg
074-1002
HRP-labeled Goat Anti-Human IgG (γ)
1.0 mg
074-1003
HRP-labeled Goat Anti-Human IgM (μ)
1.0 mg
074-1004
HRP-labeled Goat Anti-Human IgE (ε)
1.0 mg
074-1006
HRP-labeled Goat Anti-Human IgG (H+L)
1.0 mg
074-1007
HRP-labeled Goat Anti-Human IgA+IgG+IgM (H+L)
1.0 mg
14-10-01
HRP-labeled Goat Anti-Human IgA (α)
0.5 mg
214-1002
HRP-labeled F(ab’)2 Goat Anti-Human IgG (γ)
0.5 mg
214-1003
HRP-labeled F(ab’)2 Goat Anti-Human IgM (μ)
0.5 mg
214-1006
HRP-labeled F(ab’)2 Goat Anti-Human IgG (H+L)
0.5 mg
474-1002
HRP-labeled Goat Anti-Human IgG (γ), Liquid
1.0 mL
474-1003
HRP-labeled Goat Anti-Human IgM (μ), Liquid
1.0 mL
474-1006
HRP-labeled Goat Anti-Human IgG (H+L), Liquid
1.0 mL
•
4 CN Peroxidase Substrate
04-18-06
HRP-labeled Goat Anti-Mouse IgG (H+L), HSA
0.1 mg
•
04-18-15
HRP-labeled Goat Anti-Mouse IgG (H+L), RtSA, HSA
0.1 mg
TMB Membrane Peroxidase Substrate
04-18-18
HRP-labeled Goat Anti-Mouse IgG (H+L), RbSA, HSA
0.1 mg
074-1802
HRP-labeled Goat Anti-Mouse IgG (γ), HSA
1.0 mg
074-1803
HRP-labeled Goat Anti-Mouse IgM (μ), HSA
1.0 mg
074-1806
HRP-labeled Goat Anti-Mouse IgG (H+L), HSA
1.0 mg
074-18-061
HRP-labeled Goat Anti-Mouse IgG (H+L), XSA
1.0 mg
074-1807
HRP-labeled Goat Anti-Mouse IgA+IgG+IgM (H+L), HSA
1.0 mg
074-1809
HRP-labeled Goat Anti-Mouse IgG+IgM (H+L), HSA
1.0 mg
14-18-01
HRP-labeled Goat Anti-Mouse IgA (α), HSA
0.5 mg
214-1802
HRP-labeled F(ab’)2 Goat Anti-Mouse IgG (γ), HSA
0.5 mg
214-1806
HRP-labeled F(ab’)2 Goat Anti-Mouse IgG (H+L), HSA
0.5 mg
474-1802
HRP-labeled Goat Anti-Mouse IgG (γ), HSA, Liquid
1.0 mL
474-1806
HRP-labeled Goat Anti-Mouse IgG (H+L), HSA, Liquid
1.0 mL
074-1506
HRP-labeled Goat Anti-Rabbit IgG (H+L)
1.0 mg
074-15-061
HRP-labeled Goat Anti-Rabbit IgG (H+L), XSA
1.0 mg
074-1516
HRP-labeled Goat Anti-Rabbit IgG (H+L), HSA
1.0 mg
214-1516
HRP-labeled F(ab’)2 Goat Anti-Rabbit IgG (H+L), HSA
0.5 mg
474-1506
HRP-labeled Goat Anti-Rabbit IgG (H+L), Liquid
1.0 mL
474-1516
HRP-labeled Goat Anti-Rabbit IgG (H+L), HSA, Liquid
1.0 mL
14-30-00
HRP-labeled Streptavidin
0.5 mg
Phone: 800.638.3167
474-3000
HRP-labeled Streptavidin, Liquid, Molecular Grade
1.0 mL
Fax: 301.948.0169
Visit our website at www.kpl.com for a complete listing of HRP-labeled antibodies. To order or for more information on KPL’s protein research products, contact us at 800.638.3167 / 301.948.7755, FAX 301.948.0169 or visit us at www.kpl.com. For research use only. ©2009 KPL, Inc. All rights reserved. ABTS is a registered trademark of Roche Biochemicals. ML371-02
ELISA •
ABTS® 1- and 2-Component Microwell Peroxidase Substrates
•
SureBlueTM TMB Peroxidase Substrate
•
SureBlue ReserveTM TMB Peroxidase Substrate
•
TMB Peroxidase Substrate
Blotting
•
•
LumiGLO®Chemiluminescent Substrate LumiGLO ReserveTM Chemiluminescent Substrate
Whichever substrate you choose, enjoy the benefits of excellent signalto-noise and reproducibility.
Gaithersburg, MD 20878
www.kpl.com
ISO 9001:2008 Registered
Power Your Immunoassays
Using PhosphaGLO™
Using PhosphaGLO Reserve™
PhosphaGLO™ See More of What You’ve Been Missing! For Western blotting results that jump right out at you, take a closer look at the advantages of PhosphaGLOTM AP Substrates. To meet your needs for detection of proteins using Western blotting, KPL offers two chemiluminescent substrates designed for use with alkaline phosphatase conjugates. Both provide great sensitivity and unmatched convenience whether you are designing a new protocol or optimizing an existing one. Great Sensitivity With sensitivity in the femtogram range, PhosphaGLO ReserveTM AP Substrate is ideal for those situations where the protein of interest is expressed in low concentrations. PhosphaGLO AP Substrate is the substrate of choice for routine detection of proteins in the picogram range. Low Background Without Special Blockers Whether you use nitrocellulose or PVDF membranes, PhosphaGLO AP Substrates provide superior signal with low background. No special blockers are needed.
Convenience Both PhosphaGLO AP Substrates offer exceptional convenience: • Ready to Use! Substrates are formulated as one-component solutions; no need to mix or dilute. • Substrates are stable at 4° C for two years. • Results can be recorded on film or with a chemiluminescent imager. Simplified Assay Development Assay development with PhosphaGLO AP Substrates is facilitated by their long glow times, up to five days. Repeated exposures are possible. The substrates are provided with an easy-to-follow protocol. PhosphaGLO AP Substrates can, in most cases, be substituted for other AP chemiluminescent substrates with minimal optimization.
Power Your Immunoassays
PhosphaGLO PhosphaGLO AP Substrates - Product Information PhosphaGLO Reserve
PhosphaGLO
Femtogram
Picogram
Detection Limit Emission Duration
> 5 days
> 5 days
Stability
2 years at 4°C
2 years at 4°C
Recommended Membranes
Nitrocellulose and PVDF
Nitrocellulose and PVDF
Detection Methods
Film/Chemiluminescent Imager
Film/Chemiluminescent Imager
Comparison of AP Chemiluminescent Substrates PhosphaGLO and PhosphaGLO Reserve AP Substrates were compared to CDP-Star® Chemiluminescent Substrate to demonstrate the relative signal provided when detecting proteins at low concentrations. Each substrate was used according to the manufacturer's recommended protocol. PhosphaGLO and PhosphaGLO Reserve showed greater sensitivity as demonstrated by the detection of lower protein concentrations than CDP-Star on both PVDF and nitrocellulose membranes. In addition, the background with PhosphaGLO was equivalent to that seen with CDP-Star. As a result, signal-to-noise ratios are significantly higher with PhosphaGLO AP Substrates. PhosphaGLO Reserve AP Substrate
CDP-Star Chemiluminescent Substate
PhosphaGLO AP Substrate
Frequently Asked Questions Q: Will I need to use any special blocking agents in Western blotting? A: Special blocking agents are not needed for use with either PVDF or nitrocellulose membranes. Use of Detector Block may improve your signal-to-background ratio. We have found greater sensitivity with PVDF than with nitrocellulose. Q: How long are the substrates stable when stored at 4° C? Can they be stored frozen? Should they be stored in the dark? A: Both substrates are stable for 2 years from date of receipt when stored at 4° C. They can be stored frozen but repeated freezing and thawing may degrade the substrates. As a result, freezing is not recommended. The substrates should be stored in the dark. Exposure to light for short periods of time will not harm the substrates.
PVDF Membrane
Q: How does PhosphaGLO Reserve AP Substrate compare to premium HRP substrates such as LumiGLO ReserveTM? Nitrocellulose
Figure 1: Comparison of low-end sensitivity using PhosphaGLO Reserve AP Substrate, PhosphaGLO AP Substrate, and CDP-Star Chemiluminescent Substrate on nitrocellulose and PVDF membranes. Five-fold serial dilutions of Mouse IgG (2 ng - 3.2 pg) were separated by SDS-PAGE and transferred to the membranes. Protein was detected using a 1:1,000 dilution of biotin-labeled goat antimouse IgG, a 1:10,000 dilution of AP-labeled streptavidin and each respective substrate. CDP-Star was mixed with Nitro-Block-IITM Luminescence Enhancer before applying it to the nitrocellulose blot. Film was exposed for 10 minutes.
A: Detection limits for both products are in the femtogram range. Whereas LumiGLO Reserve will give you signal faster, PhosphaGLO Reserve allows you to capture sensitive images over a longer period of time. It gives you more versatility in your result exposures.
Ordering Information Catalog#
Description
Size
55-60-03
PhosphaGLOTM AP Substrate
30 mL
55-60-04
PhosphaGLO AP Substrate
100 mL
55-60-01
PhosphaGLO ReserveTM AP Substrate
30 mL
55-60-02
PhosphaGLO Reserve AP Substrate
100 mL Gaithersburg, MD 20878 Phone: 800.638.3167
To order or for more information on KPL's full line of protein and nucleic acid detection products, contact us at 800.638.3167 / 301.948.7755, FAX 301.948.0169 or visit us at www.kpl.com.
Fax: 301.948.0169 www.kpl.com
ISO 9001:2008 Registered PhosphaGLO, PhosphaGLO Reserve and LumiGLO Reserve are trademarks of KPL, Inc. CDP-Star Chemiluminescent Substate is a registered trademark and Nitro-Block-II is a trademark of Applied Biosystems. © 2005 KPL, Inc. All rights reserved. ML303-04
Power Your Immunoassays
AP Membrane Color Substrates See RED and PURPLE in your Western blots! BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium) is the most commonly used substrate for alkaline phosphatase (AP) detection in Western blotting due to its high sensitivity and low background staining. Traditional formulations of BCIP/NBT produce a deep purple color when reacted with AP conjugates. Now KPL’s new BCIP/INT formulation, FirePhosTM AP Membrane Substrate, forms a red precipitate, providing a choice of red or purple precipitating AP substrates to better optimize your individual assays. FirePhosTM AP Membrane Substrate BCIP/NBT Phosphatase Substrate Sensitive and reproducible
(Figure 1) , sharp band resolution and a clear image. The color produced is stable and linear over a wide dynamic range.
FirePhos AP Membrane Substrate
BCIP/NBT Substrate (1-Component)
BCIP/NBT Substrate (3-Component)
Figure 1: KPL’s substrates for alkaline phosphatase detection in Western blotting demonstrate high sensitivity and low background. A two-fold dilution series from 250 ng to 3.9 ng of mouse IgG was loaded onto a Tris-HCl gradient gel, then blotted onto nitrocellulose. The antigen was detected with ReserveAPTM Goat Anti-mouse Conjugate (KPL) followed by FirePhos AP Membrane and BCIP/NBT Substrates (1component and 3-component).
No matter which substrate you choose, you’ll achieve sensitive and reproducible detection of proteins on Western blots. Both substrates provide sensitivity in the low nanogram range with minimal background Power Your Immunoassays
ReserveAP Conjugates
TM
AP Membrane Color Substrates Non-fading
Improve your assay performance even further with ReserveAPTM-labeled Antibodies!
FirePhos and BCIP/NBT produce a bright red or purple precipitate that can be deposited on nitrocellulose and PVDF membranes. They resist fading when exposed to light. Convenient
KPL’s new ReserveAPTM Alkaline
These substrates are supplied as convenient one-component solutions that are ready to use. KPL’s BCIP/NBT is also available as a three-component solution. Both substrates are provided in several sizes to meet your needs.
Phosphatase-labeled Conjugates exhibit high potency and consistent performance in immunoassays. These conjugates are the result of advances in our conjugation chem-
Ordering Information Catalog#
Description
istry and offer high signal and low Size
background while meeting KPL’s standards for stability and repro-
FirePhosTM AP Membrane Substrate 50-81-30
FirePhos Membrane Phosphatase Substrate (1-Component)
100 mL
ducibility. They are ideal for
50-81-40
FirePhos Membrane Phosphatase Substrate (1-Component)
400 mL
demanding immunoassays that
50-81-34
FirePhos Membrane Phosphatase Substratee (1-Component)
1L
require high detection sensitivity, including ELISA, Western blotting
BCIP/NBT Phosphatase Membrane Substrate
and immunohistology. Visit
50-81-18
BCIP/NBT Membrane Phosphatase Substrate (1-Component)
100 mL
www.kpl.com for more information
50-81-07
BCIP/NBT Membrane Phosphatase Substrate (1-Component)
600 mL
on KPL’s ReserveAP conjugates.
50-81-10
BCIP/NBT Membrane Phosphatase Substrate (1-Component)
1L
50-81-00
BCIP/NBT Membrane Phosphatase Substrate (3-Component)
300 mL
Detector Block (5X)
240 mL
Membrane Blocking 71-83-00
KPL’s colorimetric membrane substrates are part of our comprehensive line of Western blottng kits and reagents. Ask about our AP chemiluminescent substrates that can significantly lower the detection limit of an assay.
Gaithersburg, MD 20878 Phone: 800.638.3167 Fax: 301.948.0169 www.kpl.com
To order or for more information, contact us at 800.638.3167 / 301.948.7755, fax 301.948.0169 or visit us at www.kpl.com.
ISO 9001:2008 Registered
FirePhos and ReserveAP are trademarks of KPL. ML348-03 For research use only. © 2007 KPL, Inc. All rights reserved. Power Your Immunoassays
ReserveAP Conjugates TM
TM
Spice up your assay with our red hot high potency ReserveAP Conjugates! KPL’s new ReserveAPTM Alkaline Phosphatase
line of AP conjugates in ELISA and outperform
(AP)-labeled antibody conjugates exhibit high
AP conjugates offered by other manufacturers.
potency and consistent performance in
Higher conjugate dilutability is also observed
immunoassays. These conjugates are the result
without loss of linearity, enabling precious
of advances in our conjugation technology and
antigen or primary antibody conservation.
offer higher signal than our current line of AP
Consistent Performance
conjugates while meeting the same standards for low background, stability and reproducibili-
Reproducible antibody conjugation and consis-
ty. They are intended for demanding
tent performance are verified according to our
immunoassays that require high detection sen-
ISO 9001:2008-registered quality management
sitivity, including ELISA, Western blotting and
system. Lot consistency studies in which three
immunohistology.
lots were studied by ELISA indicated minimal variability.
Higher Potency Excellent Value ReserveAP Conjugates are affinity purified and conjugated to the highest grade of alkaline
ReserveAP Conjugates provide high perform-
phosphatase. In our studies, they generate two-
ance at an economical price.
to-three fold higher values than our current Power Your Immunoassays
AP Substrates Chemiluminescent ELISA: A constant amount of antigen (mouse IgG) was coated onto a microtiter plate. The plate was probed with varying concentrations of goat anti-mouse IgG (H+L) AP conjugate, including KPL’s standard AP conjugate and ReserveAP™ conjugate, followed by KPL’s PhosphaGLO™ Chemiluminescent AP Substrate.
KPL offers a range of sensitive substrates for the detection and quantification of phosphatase (AP) activity. They provide a choice of intense colors for ELISA and blotting applications.
ELISA •
Conclusion: The results indicate that ReserveAP™ conjugate offers superior performance to our standard AP conjugate with a ~3-fold lower amount of conjugate required for detection.
ReserveAPTM Conjugates Ordering Information
•
•
Catalog#
Description
Size
0751-1001
Goat Anti-Human IgA (α)
1.0 mg
0751-1002
Goat Anti-Human IgG (γ)
1.0 mg
0751-1003 0751-1004
Goat Anti-Human IgM (µ) Goat Anti-Human IgE (ε)
0751-1006
FirePhosTM AP Microwell Substrate BluePhos® AP Microwell Substrate pNPP Phosphatase Substrate
Blotting •
1.0 mg 1.0 mg
FirePhos AP Membrane Substrate
•
BCIP/NBT Phosphatase Substrate
Goat Anti-Human IgG (H+L)
1.0 mg
•
PhosphaGLO AP Substrate
0751-1007
Goat Anti-Human IgA+IgG+IgM (H+L)
1.0 mg
•
2151-1002
0.5 mg
4751-1002
F(ab’)2 Anti-Human IgG (γ) Goat Anti-Human IgG (γ) liquid
PhosphaGLOTM Reserve AP Substrate
4751-1003
Goat Anti-Human IgM (µ) liquid
1.0 mg
4751-1006
Goat Anti-Human IgG (H+L) liquid
1.0 mg
0751-1802
Goat Anti-Mouse IgG (γ) HSA
1.0 mg
0751-1803
Goat Anti-Mouse IgM (µ) HSA
1.0 mg
0751-1806
Goat Anti-Mouse IgG (H+L) HSA
1.0 mg
0751-1809
Goat Anti-Mouse IgG +IgM (H+L) HSA
1.0 mg
4751-1802
Goat Anti-Mouse IgG (γ) HSA, liquid
1.0 mg
4751-1806
Goat Anti-Mouse IgG (H+L) HSA, liquid
1.0 mg
151-18-01
Goat Anti-Mouse IgA (α) HSA
0.5 mg
0751-1807
Goat Anti-Mouse IgA+IgG+IgM (H+L) HSA
1.0 mg
0751-1506
Goat Anti-Rabbit IgG (H+L)
1.0 mg
0751-1516
Goat Anti-Rabbit IgG (H+L) HSA
1.0 mg
4751-1506
Goat Anti-Rabbit IgG (H+L), liquid
1.0 mg
4751-1516
Goat Anti-Rabbit IgG (H+L) HSA, liquid
1.0 mg
1.0 mg
HSA=Human Serum Adsorbed
Whichever substrate you choose, enjoy the benefits of excellent signalto-noise with higher sensitivity and lower background than that of other AP substrates. Visit www.kpl.com for more information.
Gaithersburg, MD 20878 Phone: 800.638.3167 Fax: 301.948.0169 www.kpl.com
ISO 9001:2008 Registered
Visit our website at www.kpl.com for a complete listing of ReserveAP conjugates. To order or for more information on KPL’s protein research products, contact us at 800.638.3167 / 301.948.7755, FAX 301.948.0169 or visit us at www.kpl.com. ML349-06 For research use only. ©2007 KPL, Inc. All rights reserved.
Power Your Immunoassays
Immunoassay Buffers Save preparation time with KPL’s new line of quality buffers! Lighten your work load with KPL’s new line of quality
registered quality management system. All buffers are con-
buffers. Offered as convenient liquid concentrates, they
ductivity controlled. KPL offers a variety of buffers designed
eliminate the need for time-consuming buffer preparation.
for use in general immunoassay applications such as Western
KPL buffers are manufactured and carefully controlled for
blotting, gel electrophoresis, ELISA and sample preparation.
quality and consistent performance with our ISO 9001:2008Description
1X Composition
Applications
10X Tris-Glycine Transfer Buffer
25 mM Tris, 192 mM glycine. pH 8.3
10X Tris-Glycine-SDS
25 mM Tris, 192 mM glycine, 0.1% SDS. pH 8.3
For preparing a standard Western blot transfer buffer (Towbin) and as a gel electrophoresis buffer for native Tris-glycine gels without SDS. . Running buffer for sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) of proteins.
10X Phosphate-Buffered Saline (PBS)
10 mM Phosphate,150 mM NaCl. pH 7.4
Phosphate-Buffered Tween 20, (PBST)
10 mM Phosphate, 150 mM NaCl, 0.05% Tween 20. pH 7.4
10X Tris-Buffered Saline (TBS)
50 mM Tris, 150 mM NaCl. pH 7.6
10% Tween 20
10% Tween 20
Wash buffer for general immunoassay applications and sample preparation. Where applicable, Tween 20 is a nonionic detergent that reduces nonspecific binding and protein-protein interaction during wash steps.
Nonionic detergent additive for PBS and Western blotting wash solutions. Reduces nonspecific binding and proteinprotein interactions.
See reverse side.
Power Your Immunoassays
Immunoassay Buffers
KPL Quality Buffers (cont’d) Description
1X Composition
Applications
5% SDS
5% Sodium Dodecyl Sulfate
Detergent surfactant used in preparing proteins for sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) and as an additive to transfer buffers in Western blotting. SDS increases the elution rate of proteins from the gel.
20X SSC
15 mM sodium citrate, 150 mM NaCl in DEPC water. pH 7.0
Used in a variety of molecular biology applications. Facilitates transfer of nucleic acids to membranes.
10X Dulbecco's PBS (D-PBS)
8.5 mM sodium phosphate, 1.5 mM potassium phosphate, 137 mM NaCl. pH 7.4
For sample preparation and as a wash buffer in general immunoassay, tissue, and cell culture applications. Not formulated with magnesium or calcium salts. Where applicable, Tween 20 is a non-ionic detergent that reduces nonspecific binding.
Other Immunoassay Support Reagents Available from KPL ELISA and Western Blotting Applications: • AP and HRP Conjugate Stabilizers
10X Dulbecco's with Tween 20. (D-PBST)
8.5 mM sodium phosphate, 1.5 mM potassium phosphate, 137 mM NaCl, 2.7 mM KCl. 0.05% Tween 20. pH 7.4
10X Tris-Buffered Saline with 0.5% Tween 20 (TBST)
50 mM Tris, 150 mM NaCl, 0.05% Tween 20. pH 7.6
10X Tris-Buffered Saline with 10% Tween 20 (TBST)
50 mM Tris, 150 mM NaCl, 1.0% Tween 20. pH 7.6
• 10% BSA Diluent/Blocking Solution Concentrate • Milk/Diluent Blocking Solution Concentrate • 20X Wash Solution Concentrate
As a general wash buffer in immunoassay applications. Tween 20 is a non-ionic detergent that reduces nonspecific binding. The 10% Tween 20 formulation provides a more stringent wash than standard TBST formulations.
Western Blotting Applications: • Detector Block • Biotin Wash Kit
Take the time and effort out of your buffer preparation. Choose from our line of popular buffers and enjoy the benefits - convenience, reliability and confidence - that KPL’s pretested, quality buffers bring to your research.
ELISA Applications: • Coating Solution Concentrate • Stop Solutions
Ordering Information Catalog#
Description
Size
51-10-01 51-10-02 51-11-01 51-11-02 51-13-01 51-13-02 51-14-01 51-14-02 51-17-01 51-17-02 51-12-01 51-12-02 51-20-01 50-86-05 51-15-01 51-15-02 51-16-01 51-16-02 51-18-01 51-18-02 51-19-01 51-19-02
10X Tris-Glycine Transfer Buffer
1L 5L 1L 5L 1L 5L 200 mL 1L 1L 5L 200 mL 1L 200 mL 1L 1L 5L 200 mL 1L 200 mL 1L 200 mL 1L
10X Tris-Glycine SDS Buffer 10X Phosphate-Buffered Saline (PBS) 10X Phosphate-Buffered Saline with Tween 20 (PBST) 10X Tris-Buffered Saline (TBS) 10% Tween 20 5% SDS 20X SSC 10X Dulbecco's PBS (D-PBS) 10X Dulbecco's PBS with Tween 20 (D-PBST) 10X Tris Buffered Saline with 0.5% Tween 20 (TBST) 10X Tris Buffered Saline with 10% Tween 20 (TBST)
Gaithersburg, MD 20878 Phone: 800.638.3167 Fax: 301.948.0169 www.kpl.com
ISO 9001:2008 Registered
To order or for more information on KPL’s full line of protein detection products, contact us at 800.638.3167 / 301.948.7755, fax 301.948.0169 or visit us at www.kpl.com. ©2009 KPL, Inc. All rights reserved. ML363-04
Power Your Immunoassays
Ordering Information
Catalog #
Description
Size
Protein Detector™ Western Blotting Kits Each kit includes anti-mouse and anti-rabbit conjugates, Detector Block, Wash Solution Concentrate and Substrate. Phosphatase Chromogenic 55-11-50
BCIP/NBT Western Blot Kit
2500 cm2
TMB Western Blot Kit
Peroxidase Chemiluminescent 54-12-50 LumiGLO® Western Blot Kit 54-13-50
LumiGLO ReserveTM Western Blot Kit
Description
Size
Substrates for Western Blotting Phosphatase Colorimetric Substrates 50-81-18 BCIP/NBT Substrate
100 mL
50-81-07
600 mL
BCIP/NBT Substrate TM
50-81-30
FirePhos
50-81-40
FirePhos Membrane AP Substrate
400 mL
2500 cm2
50-81-34
FirePhos Membrane AP Substrate
1000 mL
2500 cm2
Phosphatase Chemiluminescent Substrates 55-60-03 PhosphaGLOTM AP Substrate
2400 cm2
55-60-04
PhosphaGLO AP Substrate
55-60-01
PhosphaGLO Reserve AP Substrate
30 mL
55-60-02
PhosphaGLO Reserve AP Substrate
100 mL
Peroxidase Chromogenic 54-11-50
Catalog #
Related Reagents and Kits Antibody Conjugates All antibodies listed below are produced in goat. For a complete antibody listing, refer to KPL’s Product Catalog.
Membrane AP Substrate
100 mL
30 mL 100 mL
Peroxidase Chromogenic Substrates 50-77-18 TMB Membrane Substrate
100 mL
Phosphatase-labeled 475-1006 Anti-Human IgG (H+L)
50-77-03
TMB Membrane Substrate
200 mL
1.0 mL, liquid
50-73-00
4 CN Substrate
600 mL
475-1806
Anti-Mouse IgG (H+L), HSA
1.0 mL, liquid
50-73-04
4 CN Substrate
2700 mL
475-1506
Anti-Rabbit IgG (H+L)
1.0 mL, liquid
Peroxidase Chemiluminescent Substrates 54-61-02 LumiGLO Chemiluminescent Substrate
60 mL
Peroxidase-labeled 474-1006 Anti-Human IgG (H+L)
1.0 mL, liquid
54-61-00
LumiGLO Chemiluminescent Substrate
240 mL
474-1806
Anti-Mouse IgG (H+L) HSA
1.0 mL, liquid
54-61-01
LumiGLO Chemiluminescent Substrate
720 mL
474-1506
Anti-Rabbit IgG (H+L)
1.0 mL, liquid
54-71-00
LumiGLO Reserve Substrate Kit
2400 cm2
54-71-01
LumiGLO Reserve Substrate Kit
600 cm2
Biotin-labeled 16-10-06 Anti-Human IgG (H+L)
0.5 mg
176-1006
2.0 mg
Anti-Human IgG (H+L)
Labeled Streptavidin 474-3000 HRP-labeled 475-3000
AP-labeled
Assay Support Reagents 50-84-00 Coating Solution Concentrate
50 mL
54-15-01
HRPStabilizer
200 mL
1.0 mL, liquid
55-15-00
APStabilizer
200 mL
1.0 mL, liquid
50-61-00
10% BSA Diluent/Blocking Solution Concentrate
200 mL
50-82-01
Milk Diluent/Blocking Solution Concentrate
200 mL
71-83-00
DetectorTM Block (5X)
240 mL
50-63-00
Wash Solution Concentrate (20X)
800 mL
50-63-06
Biotin Wash Solution Concentrate (10X) 200 mL
60-00-50
Biodyne® B Nylon Membrane
HSA=human serum adsorbed.
To order or for more information, call KPL at 800.638.3167, 301.948.7755, Fax: 301.948.0169. www.kpl.com or contact your local sales partner.
1 roll