Shop All Western Blot Substrates and Substrate Kits

NBT Substrate Powder (nitro-blue tetrazolium chloride) Thermo Scientific™

Thermo Scientific Pierce NBT and BCIP Substrates are powders and ready-made solutions of nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate for chromogenic blot and IHC staining with alkaline phosphatase probes.

The combination of NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) yields an intense, insoluble black-purple precipitate when reacted with alkaline phosphatase, a popular enzyme conjugate for antibody probes. Pierce NBT and BCIP powders are available individually and in two convenient, ready-to-use solutions (with and without a levamisole suppressor of endogenous phosphatase activity).

Features:

AP substrate—for detection of alkaline phosphatase (alk-phos) activity on solid media, including nitrocellulose and PVDF membranes and fixed tissue samples
Chromogenic—no special equipment needed for visualization; produces purple-black precipitate that is easy to photograph
Package options—choose individual NBT and BCIP powders, or preformulated, ready-to-use solutions with or without levamisole suppressor

Highlights of Pierce NBT/BCIP 1-Step Solution (Part No. 34042):
• Best for immunoblotting applications
• Ready-to-use single component
• Sensitive with low background and minimial assay-to-assay variability

Highlights of Pierce NBT/BCIP plus Suppressor 1-Step Solution (Part No. 34070):
• Best for immunohistochemistry applications
• Ready-to-use single component
• Contains 1 mM levamisole to inhibit endogenous phosphatase activity, while yielding an intense dark purple precipitate in the presence of calf intestinal phosphatase

Related Products
1-Step™ NBT/BCIP Substrate Solution
1-Step™ NBT/BCIP plus Suppressor Substrate Solution
BCIP Substrate Powder (5-bromo-4-chloro-3'-indolyphosphate p-toluidine)

CDP-Star™ Substrate (0.25 mM Ready-To-Use) Invitrogen™

The CDP-Star® Substrate is a chemiluminescent alkaline phosphatase substrate, ready-to-use for protein or nucleic acid blotting on nitrocellulose membranes. This substrate can also be used in solution-based assays.

This CDP-Star® chemiluminescent substrate for alkaline phosphatase let you detect alkaline phosphatase and alkaline phosphatase-labeled molecules with unparalleled sensitivity, speed, and ease.

• Chemiluminescent substrate provides highly sensitive replacements for the widely used fluorogenic substrate methylumbelliferyl phosphate (MUP), and the colorimetric substrate p-nitrophenyl phosphate (pNPP).
• Low background luminescence coupled with high-intensity light output enables detection of alkaline phosphatase labels with the highest possible sensitivity and signal-to-noise.

This versatile chemiluminescent substrate exhibits high sensitivity in membrane-based applications such as Southern, Northern, and Western blotting. This substrate can also be used in solution-based assays such as immunoassays, DNA probe assays, enzyme assays, and reporter gene assays. Maximum light levels are reached in approximately 10 minutes and glow emission persists for several hours.

For Research Use Only. Not for use in diagnostics procedures.

BCIP Substrate Powder (5-bromo-4-chloro-3'-indolyphosphate p-toluidine) Thermo Scientific™

Thermo Scientific Pierce NBT and BCIP Substrates are powders and ready-made solutions of nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate for chromogenic blot and IHC staining with alkaline phosphatase probes.

The combination of NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) yields an intense, insoluble black-purple precipitate when reacted with alkaline phosphatase, a popular enzyme conjugate for antibody probes. Pierce NBT and BCIP powders are available individually and in two convenient, ready-to-use solutions (with and without a levamisole suppressor of endogenous phosphatase activity).

Features:

AP substrate—for detection of alkaline phosphatase (alk-phos) activity on solid media, including nitrocellulose and PVDF membranes and fixed tissue samples
Chromogenic—no special equipment needed for visualization; produces purple-black precipitate that is easy to photograph
Package options—choose individual NBT and BCIP powders, or preformulated, ready-to-use solutions with or without levamisole suppressor

Highlights of Pierce NBT/BCIP 1-Step Solution (Part No. 34042):
• Best for immunoblotting applications
• Ready-to-use single component
• Sensitive with low background and minimial assay-to-assay variability

Highlights of Pierce NBT/BCIP plus Suppressor 1-Step Solution (Part No. 34070):
• Best for immunohistochemistry applications
• Ready-to-use single component
• Contains 1 mM levamisole to inhibit endogenous phosphatase activity, while yielding an intense dark purple precipitate in the presence of calf intestinal phosphatase

Related Products
1-Step™ NBT/BCIP Substrate Solution
1-Step™ NBT/BCIP plus Suppressor Substrate Solution
NBT Substrate Powder (nitro-blue tetrazolium chloride)

1-Step™ NBT/BCIP Substrate Solution Thermo Scientific™

Thermo Scientific Pierce NBT and BCIP Substrates are powders and ready-made solutions of nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate for chromogenic blot and IHC staining with alkaline phosphatase probes.

The combination of NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) yields an intense, insoluble black-purple precipitate when reacted with alkaline phosphatase, a popular enzyme conjugate for antibody probes. Pierce NBT and BCIP powders are available individually and in two convenient, ready-to-use solutions (with and without a levamisole suppressor of endogenous phosphatase activity).

Features:

AP substrate—for detection of alkaline phosphatase (alk-phos) activity on solid media, including nitrocellulose and PVDF membranes and fixed tissue samples
Chromogenic—no special equipment needed for visualization; produces purple-black precipitate that is easy to photograph
Package options—choose individual NBT and BCIP powders, or preformulated, ready-to-use solutions with or without levamisole suppressor

Highlights of Pierce NBT/BCIP 1-Step Solution (Part No. 34042):
• Best for immunoblotting applications
• Ready-to-use single component
• Sensitive with low background and minimial assay-to-assay variability

Highlights of Pierce NBT/BCIP plus Suppressor 1-Step Solution (Part No. 34070):
• Best for immunohistochemistry applications
• Ready-to-use single component
• Contains 1 mM levamisole to inhibit endogenous phosphatase activity, while yielding an intense dark purple precipitate in the presence of calf intestinal phosphatase

Related Products
1-Step™ NBT/BCIP plus Suppressor Substrate Solution
NBT Substrate Powder (nitro-blue tetrazolium chloride)
BCIP Substrate Powder (5-bromo-4-chloro-3'-indolyphosphate p-toluidine)

SuperSignal™ Western Blot Substrate Bundle, Atto + trial-size Pico PLUS Thermo Scientific™

This substrate bundle contains SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate plus a sample size of SuperSignal West Pico PLUS Chemiluminescent Substrate to provide chemiluminescent detection substrates for both routine and high sensitivity western blotting.

SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate
Thermo Scientific SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate is an ultra-sensitive enhanced chemiluminescent (ECL) substrate for high attogram protein level detection by western blot analysis with horseradish peroxidase (HRP) enzyme. It is designed for detection of very low-abundant targets or the maximum levels of sensitivity required when using precious samples or costly antibodies.

• Sensitivity: low-femtogram to high-attogram
• Stability: eight hours for working solution, one year for kit at 4°C
• Compatibility: nitrocellulose and PVDF membranes
• Signal duration: up to four hours
• Recommended primary antibody concentration: 1:1,000–1:5,000 dilution (0.2–1.0 µg/mL)
• Recommended secondary antibody concentration: 1:100,000–1:250,000 dilution (4–10 ng/mL)

View full product information ›

SuperSignal West Pico PLUS Chemiluminescent Substrate
Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate enables picogram to high femtogram-level protein detection by western blot analysis. It is compatible with different membranes, blocking reagents, and a wide range of antibody dilutions, making it an ideal choice for routine western blotting applications.

• Sensitivity: picogram to femtogram
• Stability: eight hours for working solution; one year for kit at room temperature
• Compatibility: nitrocellulose and PVDF membranes
• Signal duration: 6 to 24 hours
• Recommended primary antibody concentration: 1:1,000–1:5,000 dilution (0.2–1.0 µg/mL)
• Recommended secondary antibody concentration: 1:20,000–100,000 dilution (10–50 ng/mL)

View full product information ›

1-Step™ Chloronaphthol Substrate Solution Thermo Scientific™

Thermo Scientific Pierce 1-Step Chloronaphthol Solution is a solution of 4-chloro-1-naphthol for the chromogenic detection of horseradish peroxidase (HRP) activity in Western blot and tissue staining methods.

Chloronaphthol (4-CN, MW = 178.6) reacts with HRP to form a distinct blue to blue-purple product to localize and visualize the activity of peroxidase-conjugated antibodies on blots or fixed tissue samples. The HRP substrate is not as sensitive or stable as other chromogenic substrates, such as TMB and DAB. However, the blue-purple precipitate photographs well, and its alcohol-solubility is a property that can be utilized in certain double-staining applications.

Features of Chloronaphthol Substrates:

HRP substrate—for detection of horseradish peroxidase activity on solid media
Chromogenic—no special equipment needed for visualization
Blue-purple—easy to photograph in color or black-and-white
Package options—choose powder or ready-to-use solution

Highlights of Pierce CN Solution
Ready-to-use—no ethanol or methanol required to dissolve; no dilution necessary for use
Complete—does not require buffer or addition of hydrogen peroxide
Pre-filtered—does not need to be filtered before use like powders or tablets after they are dissolved
Stable—store refrigerated at 4°C for at least one year

Related Products
Chloronaphthol Substrate Powder (4-chloro-1-naphthol)

Pierce™ ECL Western Blotting Substrate Thermo Scientific™

Thermo Scientific Pierce ECL Western Blotting Substrate is a value-priced, entry-level horseradish peroxidase (HRP) substrate for enhanced chemiluminescence (ECL) that directly replaces costlier products without the need to re-optimize conditions.

Pierce ECL Plus Western Blotting Substrate characteristics include:
Sensitivity: low picogram
Stability: 1-hr working solution stability, 1-year kit stability at 4°C
Compatibility: nitrocellulose and PVDF membranes
Signal duration: 1 to 2 hours
Recommended primary antibody concentration: 1:1,000–1:5,000 dilution (0.2–10 µg/mL)
Recommended secondary antibody concentration: 1:1,000–1:15,000 dilution (0.07–1.0 µg/mL)

Compare and learn more about our western blot chemiluminescent substrates ›
View all HRP substrates available ›

Pierce ECL Western Blotting Substrate provides reliability and performance equivalent to other standard ECL substrates for detection of HRP enzyme activity. It enables the detection of picogram amounts of antigen and allows for easy detection of HRP using photographic or other imaging methods.

Because the luminol and peroxide reagent formulations are identical to other commercially available substrate products, one can switch to Pierce ECL without needing to optimize probing conditions or incubation protocols. Pierce ECL Western Blotting Substrate provides performance identical to the original Amersham ECL Western Blotting Detection Reagent from GE Healthcare.

Pierce™ CN/DAB Substrate Kit Thermo Scientific™

Thermo Scientific Pierce CN/DAB Substrate Kit includes substrate and peroxide solutions for combined chloronaphthol- and diaminobenzidine-based detection of horseradish peroxidase (HRP) activity in Western blot and tissue staining methods.

This kit combines two popular chromogenic peroxidase substrate compounds (4-CN and DAB) in a single, stable, 10X stock solution. This CN/DAB solution produces an intense dark black precipitate at sites of bound HRP-conjugated antibodies on probed blots and tissue samples. The Pierce CN/DAB Substrate Kit can be used in Western immunoblotting techniques as well as in dot blotting assays. The kit includes the 10X solution of CN/DAB and accompanying 1X Stable Peroxide Substrate Buffer.

Features of the CN/DAB Substrate Kit:

HRP substrate—for detection of horseradish peroxidase activity on solid media via the combined action of CN (4-chloro-1-naphthol) and DAB (3,3'-diaminobenzidine tetrahydrochloride)
Chromogenic—no special equipment needed for visualization; yields intense black bands that are easy to photograph
Easy-to-use kit—substrate stock solution and stable peroxide substrate buffer mean that there's no dry powders to measure and dissolve
Stable—store refrigerated at 4°C for at least one year

Includes:
• CN/DAB Solution (10X) and Stable Peroxide Substrate Solution (1X)

Prionics™-Check WESTERN Kit (12000) Thermo Scientific™

The Prionics®-Check WESTERN Kit is the gold standard of rapid BSE testing and is used as a confirmatory test by most National Reference Laboratories across Europe. It owes its success to its precision and reliability. The test uses the relative protease resistance of the disease-specific prion protein PrPSc for TSE detection. Protease digestion leads to a small decrease in PrPSc protein size, whereas the normal protein PrPC is completely degraded.

The Prionics®-Check WESTERN Kit delivers a completely reliable test-result based on three independent and easily interpretable criteria: signal (PrPSc), characteristic three-band pattern BSE positive, position relative to control (PrPC).

Test procedure
1. The homogenized brain sample is incubated with an optimized reagent mixture consisting of digestion enzymes and a buffer solution. This solution degrades the normal prion protein. Only the BSE-specific prion protein remains in the test sample.
2. The proteins in the sample are then separated according to size by gel electrophoresis and transferred to a special blot membrane for detection. The BSE-specific prion protein on the membrane is detected with specific antibodies and is visualized on a digital file or on a film.

1-Step™ Ultra TMB-Blotting Solution Thermo Scientific™

Thermo Scientific 1-Step Ultra TMB-Blotting Solution is an enhanced single-component horseradish peroxidase (HRP) substrate for Western blotting.

This precipitating, colorimetric Western blot substrate for HRP provides high sensitivity, increased signal-to-noise ratio and low background compared to many other chromogenic substrates. The blotting solution contains soluble TMB (3,3',5,5'-tetramethylbenzidine), which reacts very quickly with horseradish peroxidase enzyme to produce an insoluble dark blue precipitate that does not fade or flake. The substrate is compatible with both nitrocellulose and PVDF membranes. The blotting solution is supplied ready to use with no mixing required.

Features of 1-Step Ultra TMB-Blotting Solution:

Fast—protein bands visible in less than one minute
Fade-resistant—protein bands stable after membrane drying
Sensitive—detection limit similar to Pierce ECL Western Blotting Substrate
Chromogenic—no special equipment needed for visualization; produces dark blue bands
Ready to use—no organic solvents are required to dissolve; no dilution necessary for use

Applications:
• Chromogenic Western blot detection
• Nitrocellulose membrane blots
• PVDF membrane blots

Pierce™ DAB Substrate Kit Thermo Scientific™

The Thermo Scientific Pierce DAB Substrate Kit enables chromogenic detection of horseradish peroxidase (HRP) activity based the action of 3,3'-diaminobenzidine (DAB) in Western blot and tissue staining methods.

DAB (3,3'-diaminobenzidine tetrahydrochloride; MW = 214.1) reacts with HRP in the presence of peroxide to yield an insoluble brown-colored product at locations where peroxidase-conjugated antibodies are bound to samples. The brown precipitate is insoluble in alcohol and other organic solvents, making it an excellent substrate for immunohistochemical staining that requires the use of traditional counterstains and mounting media. The Pierce DAB Substrate Kit includes a 10X solution of DAB and Stable Peroxide Substrate Buffer. Crystalline DAB (powder) is also available.

Features DAB Substrate:

HRP substrate—for detection of horseradish peroxidase activity on solid media, including nitrocellulose and PVDF membranes and fixed tissue samples
Chromogenic—no special equipment needed for visualization; the DAB reaction produces brown bands or spots at sites of reaction with HRP-conjugated antibodies or probes
Package options—choose powder for custom formulation with buffer and peroxide or a complete kit of preformulated solutions for immediate use

Features of the Pierce DAB Substrate Kit:

Easy-to-use—dilute 10X stock solution to 1X in provided stable peroxide buffer
Safer—preformulated DAB solution minimizes safety concerns with this chemical
Complete—includes convenient solution of stable hydrogen peroxide buffer (Part No. 34062)
Stable—store refrigerated at 4°C for at least one year; freezer storage is not necessary

DAB has been used in a variety of applications, including Western blotting, immunohistochemistry and electron microscopy. Many enhancement methods have been reported for DAB involving different buffer conditions, the addition of metal ions and post-treatment applications (Ref. 1-7; also see the Pierce Metal Enhanced DAB Substrate Kit, Part No. 34065). One disadvantage of DAB compared to other substrates is the need to use the working solution immediately because it begins a reaction process upon the addition of hydrogen peroxide. This results in increased background color of the substrate. However, the brown bands resulting from DAB reactions do not fade as quickly as the color-products of other precipitating HRP substrates.

Related Products
DAB Substrate Powder (3,3'-diaminobenzidine tetrahydrochloride)

1-Step™ NBT/BCIP plus Suppressor Substrate Solution Thermo Scientific™

Thermo Scientific Pierce NBT and BCIP Substrates are powders and ready-made solutions of nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate for chromogenic blot and IHC staining with alkaline phosphatase probes.

The combination of NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) yields an intense, insoluble black-purple precipitate when reacted with alkaline phosphatase, a popular enzyme conjugate for antibody probes. Pierce NBT and BCIP powders are available individually and in two convenient, ready-to-use solutions (with and without a levamisole suppressor of endogenous phosphatase activity).

Features:

AP substrate—for detection of alkaline phosphatase (alk-phos) activity on solid media, including nitrocellulose and PVDF membranes and fixed tissue samples
Chromogenic—no special equipment needed for visualization; produces purple-black precipitate that is easy to photograph
Package options—choose individual NBT and BCIP powders, or preformulated, ready-to-use solutions with or without levamisole suppressor

Highlights of Pierce NBT/BCIP 1-Step Solution (Part No. 34042):
• Best for immunoblotting applications
• Ready-to-use single component
• Sensitive with low background and minimial assay-to-assay variability

Highlights of Pierce NBT/BCIP plus Suppressor 1-Step Solution (Part No. 34070):
• Best for immunohistochemistry applications
• Ready-to-use single component
• Contains 1 mM levamisole to inhibit endogenous phosphatase activity, while yielding an intense dark purple precipitate in the presence of calf intestinal phosphatase

Related Products
1-Step™ NBT/BCIP Substrate Solution
NBT Substrate Powder (nitro-blue tetrazolium chloride)
BCIP Substrate Powder (5-bromo-4-chloro-3'-indolyphosphate p-toluidine)

SuperSignal™ Western Blot Substrate Trial Pack, Atto & Pico PLUS Thermo Scientific™

This substrate trial pack contains sample sizes of two substrates: SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate and SuperSignal West Pico PLUS Chemiluminescent Substrate to enable trial use of the most recommended substrates for routine and high-sensitivity western blotting.

SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate
Thermo Scientific SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate is an ultra-sensitive enhanced chemiluminescent (ECL) substrate for high attogram protein level detection by western blot analysis with horseradish peroxidase (HRP) enzyme. It is designed for detection of very low-abundant targets or the maximum levels of sensitivity required when using precious samples or costly antibodies.

• Sensitivity: low-femtogram to high-attogram
• Stability: eight hours for working solution, one year for kit at 4°C
• Compatibility: nitrocellulose and PVDF membranes
• Signal duration: up to four hours
• Recommended primary antibody concentration: 1:1,000–1:5,000 dilution (0.2–1.0 µg/mL)
• Recommended secondary antibody concentration: 1:100,000–1:250,000 dilution (4–10 ng/mL)

View full product information ›

SuperSignal West Pico PLUS Chemiluminescent Substrate
Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate enables picogram to high femtogram-level protein detection by western blot analysis. It is compatible with different membranes, blocking reagents, and a wide range of antibody dilutions, making it an ideal choice for routine western blotting applications.

• Sensitivity: picogram to femtogram
• Stability: eight hours for working solution; one year for kit at room temperature
• Compatibility: nitrocellulose and PVDF membranes
• Signal duration: 6 to 24 hours
• Recommended primary antibody concentration: 1:1,000–1:5,000 dilution (0.2–1.0 µg/mL)
• Recommended secondary antibody concentration: 1:20,000–100,000 dilution (10–50 ng/mL)

View full product information ›

SuperSignal™ West Atto Ultimate Sensitivity Substrate Thermo Scientific™

SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate is an ultra-sensitive enhanced chemiluminescent (ECL) substrate for high attogram protein detection. SuperSignal West Atto substrate provides the highest-level sensitivity and better signal-to–noise ratios compared to other commercially available high-performance horseradish peroxidase (HRP) substrates.

SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate characteristics include:
• Sensitivity: low-femtogram to high-attogram
• Stability: 48 hours for working solution, one year for kit at 4°C
• Compatibility: nitrocellulose and PVDF membranes
• Signal duration: up to six hours
• Recommended primary antibody concentration: 1:1,000–1:5,000 dilution (0.2–1.0 µg/mL)
• Recommended secondary antibody concentration: 1:100,000–1:250,000 dilution (4–10 ng/mL)

Compare and learn more about our western blot chemiluminescent substrates ›
View all HRP substrates available ›

SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate is designed for superior sensitivity and signal-to–noise ratios for very low abundant targets or precious samples in western blot analysis. When combined with optimized antibody concentrations and blocking buffers, SuperSignal West Atto substrate enables detection of target proteins in amounts that are too small to be seen with typical high performance ECL substrates.

SuperSignal™ West Dura Extended Duration Substrate Thermo Scientific™

Thermo Scientific SuperSignal West Dura Extended Duration Substrate is a luminol-based enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate with stable light output for mid-femtogram level detection for western blot analysis.

SuperSignal West Dura Extended Duration Substrate characteristics include:
Sensitivity: mid-femtogram
Stability: 24-hr working solution stability; 1-year kit stability at room temperature
Compatibility: nitrocellulose and PVDF membranes
Signal duration: 24 hours
Recommended primary antibody concentration: 1:1,000–1:50,000 dilution (0.02–1.0 µg/mL)
Recommended secondary antibody concentration: 1:50,000–1:250,000 dilution (4–20 ng/mL)

Compare and learn more about our western blot chemiluminescent substrates ›
View all HRP substrates available ›

SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity and long signal duration, making it ideal for cooled CCD camera-based detection systems. Unlike substrates with signals that decline to barely detectable levels in 30–60 minutes, the signal produced with SuperSignal West Dura chemiluminescent substrate is stable for 24 hours, allowing multiple film or camera exposures.

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