Western Blotting - KPL

Western Blotting 

Some protein detection kits 

make you wonder if your eyes 

are playing tricks on you. 

SEE MORE with KPL!


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 LumiGLO ® PhosphaGLO PhosphaGLO TMB HRP 4 CN BCIP/NBT FirePhos 

Reserve HRP HRP Reserve AP AP Substrate Substrate Substrate AP Substrate Membrane 

Substrate Substrate Substrate AP Substrate 

Type chemi. chemi. chemi. chemi. color. color. color. color. 

Format 2-comp 2-comp 1-comp 1-comp 1- or 3- 2-comp 1- or 3- 1-comp 

comp 

comp 

Detection film or image film or image film or image film or image visual-dark blue visual - purple visual - purple visual - red 

Method analysis analysis analysis analysis precipitate precipitate precipitate 

Detection Sub-picogram; 0.6 picogram femtogram picogram 50 picogram 500 picogram low nanogram low nanogram 

Limit 

femtogram 

Stability of 8 hours 24 hours 2 years 2 years 1-comp- 1 hour 1-comp- NA 

Working Solution at 4 o C at 4 o C 1 year 1 year 

at 4 o C. at 4 o C. 

3-comp- 

3-comp- 

24 hours 1 hour 

Duration of 4 - 8 hours 1 - 2 hours 5 days 5 days NA NA NA NA 

Signal 

Enzyme HRP HRP AP AP HRP HRP AP AP 

Catalyst 

Kinetics 5 minutes 5 minutes 15 minutes 15 minutes 10 minutes 10 minutes 10 minutes 10 minutes 

Recommended nitrocellulose nitrocellulose nitrocellulose nitrocellulose nitrocellulose nitrocellulose nitrocellulose nitrocellulose 

Membrane or PVDF or PVDF or PVDF or PVDF or PVDF or PVDF or PVDF or PVDF 

comp = component • chemi. = chemiluminescent • color. = colorimetric • HRP = peroxidase • AP = phosphatase • NA = not applicable 

www.kpl.com

With KPL’s Protein Detector 

Western Blotting Systems, the 

results jump right out at you.


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 Reserve TM 

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 

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. 

Superior Signal to Noise 

LumiGLO Reserve delivers lower nonspecific 

signal than competitor substrates 

in its class (Figures 2 and 3). 

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-tonoise 

ratio; very low background can be 

achieved without compromise to signal 

intensity (Figures 2 and 3). Detector 

Block is also offered separately. 

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 

LumiGLO 


ECL Plus 

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. 


A B C 

ECL Plus 

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. 

www.kpl.com

LumiGLO vs. Leading Traditional Chemiluminescent Substrates 

Densitometry for LumiGLO vs. ECL 

Superior Signal to Noise with Detector Block 

50000 

45000 

40000 

LumiGLO 

ECL 

Relative Image Density 

35000 

30000 

25000 

20000 

15000 

LumiGLO Chemiluminescent 

Peroxidase Substrate 

Supersignal ® West Pico 

10000 

5000 

0 

0 50 100 150 200 250 

Concentration (ng) 

ECL Plus 

ECL 

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. 

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. 

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. 

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. 

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. 

LumiGLO Western Blot Kit 

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-tonoise ratio 

(Figure 5). 



Sensitivity and Convenience with 

PhosphaGLO TM AP Substrates 

KPL offers two chemiluminescent substrates 

for use with alkaline phosphatase, 

PhosphaGLO Reserve TM and PhosphaGLO TM 

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 

4 o C. Their long glow times of 5 days 

facilitate assay development, enabling 

repeat exposures. 

Colorimetric Kits 

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. 

Protein Detector TM TMB 


The Protein Detector TM 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. 

Protein Detector TM BCIP/NBT 


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. 

Western Blot Reagents 

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. 

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. 

PhosphaGLO Reserve PhosphaGLO CDP Star ® 

AP Substrate AP Substrate 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. 


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. 

(Figure 1) , sharp band resolution and a clear image. 

The color produced is stable and linear over a wide 

dynamic range. 

Now KPL’s new BCIP/INT formulation, FirePhos TM 

AP Membrane Substrate, forms a red precipitate, providing 

a choice of red or purple precipitating AP substrates 

to better optimize your individual assays. 

FirePhos TM AP Membrane Substrate 

BCIP/NBT Phosphatase Substrate 

Sensitive and reproducible 

FirePhos AP BCIP/NBT Substrate BCIP/NBT Substrate 

Membrane Substrate (1-Component) (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 ReserveAP TM 

Goat Anti-mouse Conjugate (KPL) followed by 

FirePhos AP Membrane and BCIP/NBT Substrates (1- 

component 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 


AP Membrane Color Substrates 

ReserveAP TM 

Conjugates 

Non-fading 

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 

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. 

Improve your assay 

performance even further 

with ReserveAP TM -labeled 

Antibodies! 

KPL’s new ReserveAP TM Alkaline 

Phosphatase-labeled Conjugates 

exhibit high potency and consistent 

performance in immunoassays. 

These conjugates are the result of 

Ordering Information 

Catalog# Description Size 

FirePhos TM AP Membrane Substrate 

50-81-30 FirePhos Membrane Phosphatase Substrate (1-Component) 100 mL 

50-81-40 FirePhos Membrane Phosphatase Substrate (1-Component) 400 mL 

50-81-34 FirePhos Membrane Phosphatase Substratee (1-Component) 1 L 

BCIP/NBT Phosphatase Membrane Substrate 

50-81-18 BCIP/NBT Membrane Phosphatase Substrate (1-Component) 100 mL 


50-81-10 BCIP/NBT Membrane Phosphatase Substrate (1-Component) 1 L 

advances in our conjugation chemistry 

and offer high signal and low 

background while meeting KPL’s 

standards for stability and reproducibility. 

They are ideal for 

demanding immunoassays that 

require high detection sensitivity, 

including ELISA, Western blotting 

and immunohistology. Visit 

www.kpl.com for more information 

on KPL’s ReserveAP conjugates. 


Membrane Blocking 

71-83-00 Detector Block (5X) 240 mL 

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. 

SEE MORE with KPL! 

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. 

Gaithersburg, MD 20878 

Phone: 800.638.3167 

Fax: 301.948.0169 

www.kpl.com 

ISO 9001:2000 Registered 

FirePhos and ReserveAP are trademarks of KPL. 

ML348-02 

For research use only. 

© 2007 KPL, Inc. All rights reserved. 


Sensitivity, reliability and affordability! 

HRP Membrane Color Substrates 

KPL offers two substrates commonly used to detect 

horseradish peroxidase (HRP) conjugated antibodies 

that bring complementary benefits to membrane 

detection; TMB (3, 3´, 5, 5´-tetramethylbenzidene) 

Membrane Substrate and 4 CN (4-chloro-1-naphthol) 

Membrane Substrate. TMB substrate offers the most 

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 

background. 

• TMB Membrane Substrates (1- and 

(3-Component) 

In the presence of peroxidase conjugate, KPL’s TMB 

Membrane Substrates produce a stable, dark blue precipitate 

at the site of a positive reaction, enabling 

detection of as little as 50 pg of peroxidase. Our TMB 

substrates are available as 1- and 3- component solutions. 

1-component TMB Peroxidase Substrate is ready 

to use and contains hydrogen peroxide and buffer. 

Our 3-component TMB contains TMB in an organic 

base, H 2 O 2 in a citric acid buffer and a third component, 

TMB Membrane Enhancer. It provides sensitivity 

equivalent to 1-component TMB at an economic 

price. The substrate may be adapted as a soluble substrate 

for ELISA by omitting TMB Membrane 

Enhancer. Both substrates perform equally well on 

nitrocellulose and PVDF membranes. 

4 CN TMB 

4 CN produces a 

distinct, blue-purple 

precipitate and 

extremely low 

background. 

TMB produces a 

blue precipitate 

and provides 

greater sensitivity 

than 4 CN. 

• 4 CN Peroxidase Substrate (2-Component) 

4 CN (4-chloro-1-napthol) Peroxidase Membrane 

Substrate produces a purple precipitate at the reaction 

site in the presence of peroxidase conjugates. Due to 

its low background, 4 CN presents a desirable alternative 

to other, more sensitive substrates when background 

poses a problem. 


Support Reagents 

Benefits of KPL’s HRP Membrane Substrates 

• 

• 

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 

• Easy mix-and-use, two-component solutions 

• Very low background, moderate sensitivity 

• High contrast image ideal for photography and publication 


50-77-18 TMB Membrane Substrate (1-Component) 100 mL 


50-77-04 TMB Membrane Substrate (1-Component) 1 L 


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. 

• Detector TM Block 

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. 

50-77-01 TMB Membrane Enhancer 40 mL 

50-73-00 4 CN Substrate System (2-Component) 600 mL 

50-73-04 4 CN Substrate System (2-Component) 2700 mL 

Protein Detector TMB Western Blot Kit 

54-11-50 Protein Detector TMB Western Blot Kit 2500 cm 2 

Detector Block 

71-83-00 Detector Block 240 mL 

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. 


Phone: 800.638.3167 

Fax: 301.948.0169 

www.kpl.com 


Protein Detector is a trademark of KPL. 

©2008 KPL, Inc. All rights reserved. 

ML353-01 


Immunoassay Buffers 

Save preparation time with KPL’s new line of quality buffers! 

Lighten your work load with KPL’s new line of quality 

buffers. Offered as convenient liquid concentrates, they 

eliminate the need for time-consuming buffer preparation. 

KPL buffers are manufactured and carefully controlled for 

quality and consistent performance with our ISO 9001:2008- 

registered quality management system. All buffers are conductivity 

controlled. KPL offers a variety of buffers designed 

for use in general immunoassay applications such as Western 

blotting, gel electrophoresis, ELISA and sample preparation. 

Description 1X Composition Applications 

10X Tris-Glycine 25 mM Tris, 192 mM glycine. For preparing a standard Western blot transfer buffer 

Transfer Buffer pH 8.3 (Towbin) and as a gel electrophoresis buffer for native 

Tris-glycine gels without SDS. 

. 

10X Tris-Glycine-SDS 25 mM Tris, 192 mM glycine, Running buffer for sodium dodecyl sulfate – polyacrylamide 

0.1% SDS. pH 8.3 gel electrophoresis (SDS-PAGE) of proteins. 

10X Phosphate-Buffered 10 mM Phosphate,150 mM NaCl. Wash buffer for general immunoassay applications 

Saline (PBS) pH 7.4 and sample preparation. Where applicable, Tween 20 

is a nonionic detergent that reduces nonspecific 

Phosphate-Buffered 10 mM Phosphate, 150 mM NaCl, binding and protein-protein interaction during wash steps. 

Tween 20, (PBST) 0.05% Tween 20. pH 7.4 

10X Tris-Buffered Saline 50 mM Tris, 150 mM NaCl. 

(TBS) pH 7.6 

10% Tween 20 10% Tween 20 Nonionic detergent additive for PBS and Western blotting 

wash solutions. Reduces nonspecific binding and proteinprotein 

interactions. 

See reverse side. 


KPL Quality Buffers (cont’d) 

Description 1X Composition Applications 

5% SDS 5% Sodium Detergent surfactant used in preparing proteins for 

Dodecyl Sulfate 

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, Used in a variety of molecular biology applications. 

150 mM NaCl Facilitates transfer of nucleic acids to membranes. 

in DEPC water. pH 7.0 

10X Dulbecco's 8.5 mM sodium phosphate, For sample preparation and as a wash buffer in general 

PBS (D-PBS) 1.5 mM potassium phosphate, immunoassay, tissue, and cell culture applications. 

137 mM NaCl. pH 7.4 Not formulated with magnesium or calcium salts. Where 

applicable, Tween 20 is a non-ionic detergent that reduces 

nonspecific binding. 

10X Dulbecco's 8.5 mM sodium phosphate, 

with Tween 20. 

(D-PBST) 


1.5 mM potassium phosphate, 

137 mM NaCl, 2.7 mM KCl. 

0.05% Tween 20. pH 7.4 

10X Tris-Buffered 50 mM Tris, 150 mM NaCl, As a general wash buffer in immunoassay applications. 

Saline with 0.5% 0.05% Tween 20. pH 7.6 Tween 20 is a non-ionic detergent that reduces nonspecific 

Tween 20 (TBST) 

binding. The 10% Tween 20 formulation provides a more 

stringent wash than standard TBST formulations. 

10X Tris-Buffered 50 mM Tris, 150 mM NaCl, 

Saline with 10% 1.0% Tween 20. pH 7.6 

Tween 20 (TBST) 

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. 

Immunoassay 

Buffers 

Other Immunoassay Support 

Reagents Available from KPL 

ELISA and Western Blotting 

Applications: 

• AP and HRP Conjugate Stabilizers 

• 10% BSA Diluent/Blocking Solution 

Concentrate 

• Milk/Diluent Blocking Solution 

Concentrate 

• 20X Wash Solution Concentrate 

Western Blotting Applications: 

• Detector Block 

• Biotin Wash Kit 

ELISA Applications: 

• Coating Solution Concentrate 

• Stop Solutions 


51-10-01 10X Tris-Glycine Transfer Buffer 1 L 

51-10-02 5 L 

51-11-01 10X Tris-Glycine SDS Buffer 1 L 

51-11-02 5 L 

51-13-01 10X Phosphate-Buffered Saline (PBS) 1 L 

51-13-02 5 L 

51-14-01 10X Phosphate-Buffered Saline with 200 mL 

51-14-02 Tween 20 (PBST) 1 L 

51-17-01 10X Tris-Buffered Saline (TBS) 1 L 

51-17-02 5 L 

51-12-01 10% Tween 20 200 mL 

51-12-02 1 L 

51-20-01 5% SDS 200 mL 

50-86-05 20X SSC 1 L 

51-15-01 10X Dulbecco's PBS (D-PBS) 1 L 

51-15-02 5 L 

51-16-01 10X Dulbecco's PBS with Tween 20 (D-PBST) 200 mL 

51-16-02 1 L 

51-18-01 10X Tris Buffered Saline with 0.5% Tween 20 200 mL 

51-18-02 (TBST) 1 L 

51-19-01 10X Tris Buffered Saline with 10% Tween 20 200 mL 

51-19-02 (TBST) 1 L 

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. 


Phone: 800.638.3167 

Fax: 301.948.0169 

www.kpl.com 



ML363-03 


ReserveAP TM 

Conjugates 

Spice up your assay with our red hot high potency ReserveAP TM Conjugates! 

KPL’s new ReserveAP TM Alkaline Phosphatase 

(AP)-labeled antibody conjugates exhibit high 

potency and consistent performance in 

immunoassays. These conjugates are the result 

of advances in our conjugation technology and 

offer higher signal than our current line of AP 

conjugates while meeting the same standards 

for low background, stability and reproducibility. 

They are intended for demanding 

immunoassays that require high detection sensitivity, 

including ELISA, Western blotting and 

immunohistology. 

Higher Potency 

ReserveAP Conjugates are affinity purified and 

conjugated to the highest grade of alkaline 

phosphatase. In our studies, they generate twoto-three 

fold higher values than our current 

line of AP conjugates in ELISA and outperform 

AP conjugates offered by other manufacturers. 

Higher conjugate dilutability is also observed 

without loss of linearity, enabling precious 

antigen or primary antibody conservation. 

Consistent Performance 

Reproducible antibody conjugation and consistent 

performance are verified according to our 

ISO 9001:2000-registered quality assurance 

system. Lot consistency studies in which three 

lots were studied by ELISA indicated minimal 

variability. 

Excellent Value 

ReserveAP Conjugates provide high performance 

at an economical price. 


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 

• FirePhos TM AP Microwell 

Substrate 

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. 

ReserveAP TM Conjugates Ordering Information 


0751-1001 Goat Anti-Human IgA (α) 1.0 mg 

0751-1002 Goat Anti-Human IgG (γ) 1.0 mg 

0751-1003 Goat Anti-Human IgM (µ) 1.0 mg 

0751-1004 Goat Anti-Human IgE (ε) 1.0 mg 

0751-1006 Goat Anti-Human IgG (H+L) 1.0 mg 

0751-1007 Goat Anti-Human IgA+IgG+IgM (H+L) 1.0 mg 

2151-1002 F(ab’) 2 Anti-Human IgG (γ) 0.5 mg 

4751-1002 Goat Anti-Human IgG (γ) liquid 1.0 mg 

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 

HSA=Human Serum Adsorbed 

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. 

ML349-04 



• BluePhos ® AP Microwell 

Substrate 

• pNPP Phosphatase Substrate 

Blotting 

• FirePhos AP Membrane 

Substrate 

• BCIP/NBT Phosphatase Substrate 

• PhosphaGLO AP Substrate 

• PhosphaGLO TM Reserve AP 

Substrate 

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. 


Phone: 800.638.3167 

Fax: 301.948.0169 

www.kpl.com 



DyLight TM Fluorescent Conjugates 

KPL’s DyLight TM Conjugates - A Brilliant Choice! 

KPL’s DyLight TM 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 CyDye TM 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) 


DyLight TM 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 

Green 

Yellow 

Orange 

Red 

Red 

Near IR 

Infrared 

nm = nanometers 

IR = infrared 

DyLight 405 400/420 Alexa 405 and 

Cascade Blue 

DyLight 488 493/518 Alexa 488, Fluorescein 

and FITC 

DyLight 549 550/568 Alexa 546, Alexa 555, 

Cy3, TRITC 

DyLight 594 593/618 Alexa 594, Texas Red 

DyLight 633 638/658 Alexa 633 

DyLight 649 646/674 Alexa 647, Cy5 

DyLight 680 682/715 Alexa 680, Cy5.5 

DyLight 800 770/794 IRDye 800 


Description DyLight 405* DyLight 488 DyLight 549 DyLight 594* DyLight 633* DyLight 649 DyLight 680 DyLight 800 

Anti-Mouse IgG (γ), HSA 072-03-18-02 072-04-18-02 072-05-18-02 

Anti-Mouse IgG (H+L), HSA 072-08-18-06 072-03-18-06 072-04-18-06 072-09-18-06 072-10-18-06 072-05-18-06 072-06-18-06 072-07-18-06 

Anti-Mouse IgG (H+L), HSA, 0.1 mg 042-08-18-06 042-03-18-06 042-04-18-06 042-09-18-06 042-10-18-06 042-05-18-06 042-06-18-06 042-07-18-06 

Anti-Mouse IgG (H+L), RbSA, HSA 072-03-18-18 072-04-18-18 072-05-18-18 072-06-18-18 072-07-18-18 

Anti-Mouse IgM (µ), HSA 072-03-18-03 072-04-18-03 072-05-18-03 

Anti-Mouse IgG+IgM (H+L), HSA 072-03-18-09 072-04-18-09 072-05-18-09 

F(ab’) 2 Anti-Mouse IgG (γ), HSA 202-03-18-02 202-04-18-02 202-05-18-02 

F(ab’) 2 Anti-Mouse IgG (H+L), HSA 202-08-18-06 202-03-18-06 202-04-18-06 202-09-18-06 202-10-18-06 202-05-18-06 202-06-18-06 202-07-18-06 

Anti-Human IgG (H+L) 072-08-10-06 072-03-10-06 072-04-10-06 072-09-10-06 072-10-10-06 072-05-10-06 072-06-10-06 072-07-10-06 

Anti-Human IgG (H+L), 0.1 mg 042-08-10-06 042-03-10-06 042-04-10-06 042-09-10-06 042-10-10-06 042-05-10-06 042-06-10-06 042-07-10-06 

Anti-Human IgG (γ) 072-03-10-02 072-04-10-02 072-05-10-02 

Anti-Human IgM (µ) 072-03-10-03 072-04-10-03 072-05-10-03 

F(ab’) 2 Anti-Human IgG (γ) 202-03-10-02 202-04-10-02 202-05-10-02 

F(ab’) 2 Anti-Human IgM (µ) 202-03-10-03 202-04-10-03 202-05-10-03 

F(ab’) 2 Anti-Human IgG (H+L) 202-08-10-06 202-03-10-06 202-04-10-06 202-09-10-06 202-10-10-06 202-05-10-06 202-06-10-06 202-07-10-06 

Anti-Rabbit IgG (H+L) 072-08-15-06 072-03-15-06 072-04-15-06 072-09-15-06 072-10-15-06 072-05-15-06 072-06-15-06 072-07-15-06 

Anti-Rabbit IgG (H+L), 0.1 mg 042-08-15-06 042-03-15-06 042-04-15-06 042-09-15-06 042-10-15-06 042-05-15-06 042-06-15-06 042-07-15-06 

Anti-Rabbit IgG (H+L), HSA 072-03-15-16 072-04-15-16 072-05-15-16 072-06-15-16 072-07-15-16 

F(ab’) 2 Anti-Rabbit IgG (H+L), HSA 202-08-15-16 202-03-15-16 202-04-15-16 202-09-15-16 202-10-15-16 202-05-15-16 202-06-15-16 202-07-15-16 

Anti-Rat IgG (H+L) 072-08-16-06 072-03-16-06 072-04-16-06 072-09-16-06 072-10-16-06 072-05-16-06 072-06-16-06 072-07-16-06 

Anti-Rat IgG (H+L), 0.1 mg 042-03-16-06 

Anti-Guinea Pig IgG (H+L) 072-03-17-06 072-04-17-06 072-05-17-06 072-06-17-06 072-07-17-06 

Anti-Chicken IgG (H+L) 072-08-24-06 072-03-24-06 072-04-24-06 072-09-24-06 072-10-24-06 072-05-24-06 072-06-24-06 072-07-24-06 

Anti-Dog IgG (H+L) 072-03-19-06 072-04-19-06 072-05-19-06 072-06-19-06 072-07-19-06 

Anti-Goat IgG (H+L) 072-08-13-06 072-03-13-06 072-04-13-06 072-09-13-06 072-10-13-06 072-05-13-06 072-06-13-06 072-07-13-06 

Anti-Horse IgG (H+L) 072-03-21-06 072-04-21-06 072-05-21-06 072-06-21-06 072-07-21-06 

Rabbit Anti-Sheep IgG (H+L) 072-03-23-06 072-04-23-06 072-05-23-06 072-06-23-06 072-07-23-06 

Anti-Swine IgG (H+L) 072-03-14-06 072-04-14-06 072-05-14-06 072-06-14-06 072-07-14-06 

Streptavidin 072-08-30-00 072-03-30-00 072-04-30-00 072-09-30-00 072-10-30-00 072-05-30-00 072-06-30-00 072-07-30-00 

Streptavidin, 0.1 mg 042-08-30-00 042-03-30-00 042-04-30-00 042-09-30-00 042-10-30-00 042-05-30-00 042-06-30-00 042-07-30-00 

*Coming soon! 


Near Infrared and 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 

Near Infrared Fluorescent Imaging with Secondary 

Antibodies labeled with DyLight 680 and 800 

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). 

Rat hippocampal neurons 

WCS stained with; DyLight 488 

(green). MAP2 stained with 

DyLight 549 (red). 

DyLight TM Fluorescent 

Conjugates 

KPL Antibodies- 

What 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. 

HMVEC-L primary endothelial cells 

F-actin detected with DY554-Phalloidin 

(rendered green). Microtubules 

stained with DyLight 649 

(red). Nuclei detected with DAPI 

(blue). 

A549 cells 

Cytokeratin stained with DyLight 

680 (pseudocolored white). 

Lamin A stained with DyLight 

549 (pseudocolored red). 

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. 

NIH 3T3 cells 

F-actin detected with DY554-Phalloidin 

(rendered green). Microtubules 

stained with DyLight 649 

(red). Nuclei detected with DAPI 

(blue) 

Gaithersburg, MD 

Phone: 800.638.3167/301.948.7755 

Fax: 301.948.0169 

www.kpl.com 

To order or for more information on KPL’s line of unlabeled and conjugated 

affinity purified antibodies, contact us at 800.638.3167/301.948.7755, 

fax 301.948.0169 or visit www.kpl.com. 


ML356-02 


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. 



Cy3 and Cy5 Conjugates 

Image above: Confocal fluorescence micrograph of HeLa cells stained with monoclonal antibody against mitochondria enzyme and Cy3- 

conjugated anti-mouse antibody (red); rabbit polyclonal antibody to histones in DNA and Cy5-conjugated anti-rabbit antibody (blue). 

KPL Cy TM Dye Conjugates- Improve your assay and brighten your day! 

Cy Dyes offer intense fluorescence when coupled 

with KPL’s affinity purified antibodies and streptavidin. 

The sensitivity and reproducibility of KPL 

antibodies combined with the brightness of Cy3 and 

Cy5 dyes produce an exceptional set of conjugates 

ideal for multiple labeling experiments. Dye/protein 

ratios have been established to ensure optimal fluorescence 

with minimal background. They present 

maximum excitation/emission spectra at 550/570 

nm (Cy3) and 650/670 nm (Cy5). 

Fluorescence 

Cy3 

Wavelength (nm) 

Cy5 

Ex 

Em 

Cy dyes are excellent alternatives to most other fluorrescein 

dyes as they are brighter and offer greater 

photostability. Cy Dye conjugates are used in both 

visual and image analysis fluorescent microscopy 

and in situ hybridization. Cy3 conjugates are ideally 

used in visual color applications, whereas Cy5 conjugates 

emit in the far red spectrum and are not easily 

visualized. 

Benefits of KPL Cy Dye Conjugates 

High performance conjugates – optimized dyeprotein 

ratios ensure high signal-to-noise. 

Intense fluorescence – offers greater sensitivity 

than TRITC conjugates. 

Narrow emission spectra – enables sensitive, 

multi-color analysis. 

Excellent photostability – more photostable than 

TRITC conjugates. 

Consistent performance – minimal lot-to-lot variation 

reduces need for assay optimization. 

Buffer stability – after rehydration, conjugates are 

stable at pH 4-9. 

Instrument compatibility – excitation and emission 

spectra correspond with standard filter sets 

and laser settings. 


Excellent Performance 

As demonstrated below KPL Cy3- and Cy5-labeled conjugates produce brighter fluorescence than 

those of other suppliers. 

Cy3 and Cy5 

Conjugates 

Cytomegalovirus-infected cells 

detected with a biotinylated CMV 

probe and the DNADetector TM 

Fluorescent in situ Hybridization Kit 

using Cy3-Strept-avidin and DAPI. 

Microplates were coated with serially diluted mouse IgG at the indicated concentrations. Conjugates at a 

concentration of 0.01 mg/mL were applied and incubated for 30 minutes. Fluorescence was measured with 

a Perkin Elmer VICTOR 3 Multilabel Plate Reader. 


Description Cy3 Cy5 

Anti-Mouse IgG (γ), HSA 072-01-18-02 072-02-18-02 

F(ab’) 2 Anti-Mouse IgG (γ), HSA 202-01-18-02 202-02-18-02 

Anti-Mouse IgG (H+L), HSA 072-01-18-06 072-02-18-06 

F(ab’) 2 Anti-Mouse IgG (H+L), HSA 202-01-18-06 202-02-18-06 

Anti-Mouse IgG (H+L), RbSA, HSA 072-01-18-18 072-02-18-18 

Anti-Mouse IgM (µ), HSA 072-01-18-03 072-02-18-03 

Anti-Mouse IgG+IgM (H+L), HSA 072-01-18-09 072-02-18-09 

Anti-Rabbit IgG (H+L) 072-01-15-06 072-02-15-06 

F(ab’) 2 Anti-Rabbit IgG (H+L), HSA 202-01-15-16 202-02-15-16 

Anti-Rabbit IgG (H+L), HSA 072-01-15-16 072-02-15-16 

Anti-Rat IgG (H+L) 072-01-16-06 072-02-16-06 

F(ab’) 2 Anti-Human IgG (H+L) 202-01-10-06 202-02-10-06 

Anti-Human IgG (γ) 072-01-10-02 072-02-10-02 

F(ab’) 2 Anti-Human IgG (γ) 202-01-10-02 202-02-10-02 

Anti-Human IgM (µ) 072-01-10-03 072-02-10-03 

F(ab’) 2 Anti-Human IgM (µ) 202-01-10-03 202-02-10-03 

Anti-Guinea Pig IgG (H+L) 072-01-17-06 072-02-17-06 

Anti-Chicken IgG (H+L) 072-01-24-06 072-02-24-06 

Anti-Horse IgG (H+L) 072-01-21-06 072-02-21-06 

Anti-Swine IgG (H+L) 072-01-14-06 072-02-14-06 

Anti-Dog IgG (H+L) 072-01-19-06 072-02-19-06 

Anti-Sheep IgG (H+L) 072-01-23-06 072-02-23-06 

Anti-Goat IgG (H+L) 072-01-13-06 072-02-13-06 

Streptavidin 072-01-30-00 072-02-30-00 

HSA=human serum adsorbed RbSA=rabbit serum adsorbed 

Cy Dye antibody conjugates are made in goat except anti-goat and anti-sheep antibodies 

made in rabbit. Supplied in 1 mg lyophilized form. 

To order or for more information on KPL’s line of unlabeled and conjugated 

affinity purified antibodies, contact us at 800.638.3167 / 301.948.7755, fax 

301.948.0169 or visit us at www.kpl.com. 

Signal Detection 

Cy3 is excited maximally at 550 nm 

and fluoresces maximally at 570 nm. 

It is excited to about 50% of maximum 

with an argon laser (514 nm or 

528 nm lines), or to about 75% of 

maximum with a helium/neon laser 

(543 nm line) or mercury lamp (546 

nm line). 

Cy5 is excited maximally at 650 nm 

and fluoresces maximally at 670 nm. 

It is excited to about 98% of maximum 

with a krypton/argon laser (647 

nm line) or to about 63% of maximum 

with a helium/neon laser (633 

nm line). Cy5 produces minimal autofluorescence 

of biological specimens 

in this region of the spectrum. 

A confocal microscope equipped with 

the appropriate laser for excitation 

and a far-red detector enable double 

labeling with Cy3 and Cy5. 


Phone: 800.638.3167 

Fax: 301.948.0169 

www.kpl.com 


ML368-01 


Cy Dye is a trademark of GE Healthcare. 



Peroxidase Conjugates 

KPL Peroxidase Conjugates: Time-tested, Sensitive and Reliable 

Since 1979 KPL has provided quality affinity 

purified antibodies to researchers worldwide. 

Over the years we have refined our production 

process to provide antibodies with high potency 

and consistent performance in immunoassays. 

From the start KPL gives careful consideration 

to immunogen preparation, using a highly 

purifed formulation to generate antiserum. KPL 

pools antiserum from multiple animals to 

reduce natural animal to animal serum variability. 

During the purification process our ISO 

9001:2008-certified quality procedures impose 

rigorous standards for potency and cross-reactivity. 

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 

streptavidin. They are affinity purified and in 

some cases further adsorbed to minimize crossreactivity 

between animal species or shared 

reactivity with other immunoglobulin classes. 

HRP-labeled F(ab’) 2 fragment antibodies are 

offered for assays requiring extremely low background 

and absence of F(c)-mediated binding. 

KPL reacts HRP of the highest quality with 

affinity purified antibodies and streptavidin 

using the periodate method of Nakane and 

Kawaoi. Special features of HRP include: 

• faster catalytic rate than alkaline phosphatase 

• generates more product in shorter incubation 

times 

• provides maximum sensitivity, low nonspecific 

binding 

• ideal for ELISA, Western blotting and 

immunohistology applications. 


Peroxidase Conjugates 

Peroxidase Conjugates Ordering Information 

(Partial listing) 


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 

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 

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 

474-3000 HRP-labeled Streptavidin, Liquid, Molecular Grade 1.0 mL 

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. 

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. 

ELISA 

• ABTS ® 1- and 2-Component 

Microwell Peroxidase Substrates 

• SureBlue TM TMB Peroxidase 

Substrate 

• SureBlue Reserve TM TMB 

Peroxidase Substrate 

• TMB Peroxidase Substrate 

Blotting 

• 4 CN Peroxidase Substrate 

• TMB Membrane Peroxidase 

Substrate 

• LumiGLO ® Chemiluminescent 

Substrate 

• LumiGLO Reserve TM 

Chemiluminescent Substrate 

Whichever substrate you choose, 

enjoy the benefits of excellent signalto-noise 

and reproducibility. 


Phone: 800.638.3167 

Fax: 301.948.0169 

www.kpl.com 




ABTS is a registered trademark of Roche Biochemicals. 

ML371-01 



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 cm 2 

Peroxidase Chromogenic 

54-11-50 TMB Western Blot Kit 2500 cm 2 

Peroxidase Chemiluminescent 

54-12-50 LumiGLO ® Western Blot Kit 2500 cm 2 

54-13-50 LumiGLO Reserve TM Western 2400 cm 2 

Blot Kit 

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. 

Phosphatase-labeled 

475-1006 Anti-Human IgG (H+L) 1.0 mL, liquid 

475-1806 Anti-Mouse IgG (H+L), HSA 1.0 mL, liquid 

475-1506 Anti-Rabbit IgG (H+L) 1.0 mL, liquid 

Peroxidase-labeled 

474-1006 Anti-Human IgG (H+L) 1.0 mL, liquid 

474-1806 Anti-Mouse IgG (H+L) HSA 1.0 mL, liquid 

474-1506 Anti-Rabbit IgG (H+L) 1.0 mL, liquid 

Biotin-labeled 

16-10-06 Anti-Human IgG (H+L) 0.5 mg 

176-1006 Anti-Human IgG (H+L) 2.0 mg 

Labeled Streptavidin 

474-3000 HRP-labeled 1.0 mL, liquid 

475-3000 AP-labeled 1.0 mL, liquid 

Catalog # Description Size 

Substrates for Western Blotting 

Phosphatase Colorimetric Substrates 

50-81-18 BCIP/NBT Substrate 100 mL 

50-81-07 BCIP/NBT Substrate 600 mL 

50-81-30 FirePhos TM Membrane AP Substrate 100 mL 

50-81-40 FirePhos Membrane AP Substrate 400 mL 

50-81-34 FirePhos Membrane AP Substrate 1000 mL 

Phosphatase Chemiluminescent Substrates 

55-60-03 PhosphaGLO TM AP Substrate 30 mL 

55-60-04 PhosphaGLO AP Substrate 100 mL 

55-60-01 PhosphaGLO Reserve AP Substrate 30 mL 

55-60-02 PhosphaGLO Reserve AP Substrate 100 mL 

Peroxidase Chromogenic Substrates 

50-77-18 TMB Membrane Substrate 100 mL 

50-77-03 TMB Membrane Substrate 200 mL 

50-73-00 4 CN Substrate 600 mL 

50-73-04 4 CN Substrate 2700 mL 

Peroxidase Chemiluminescent Substrates 

54-61-02 LumiGLO Chemiluminescent Substrate 60 mL 



54-71-00 LumiGLO Reserve Substrate Kit 2400 cm 2 

54-71-01 LumiGLO Reserve Substrate Kit 600 cm 2 

Assay Support Reagents 

50-84-00 Coating Solution Concentrate 50 mL 

54-15-01 HRPStabilizer 200 mL 

55-15-00 APStabilizer 200 mL 

50-61-00 10% BSA Diluent/Blocking Solution 200 mL 

Concentrate 

50-82-01 Milk Diluent/Blocking Solution 200 mL 

Concentrate 

71-83-00 Detector TM 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 1 roll 

HSA=human serum adsorbed. 

ML282-04 

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.

Western Blotting - KPL

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