Shop All Horseradish Peroxidase (HRP) Substrates

TMB

TMB (3,3´,5,5´-tetramethylbenzidine) is the most popular chromogenic substrate for horseradish peroxidise (HRP) detection in ELISA, yielding a blue color when oxidized.

QuantaBlu™ Fluorogenic Peroxidase Substrate Kit (Thermo Scientific™)

The Thermo Scientific QuantaBlu Fluorogenic Peroxidase Substrate Kit is a three-component kit containing proprietary fluorescent substrate, peroxide and stop solutions for stopped (endpoint) or non-stopped assays. QuantaBlu Fluorogenic Peroxidase Substrates allow rapid stopped and kinetic ELISA assays to be performed with greater sensitivity than TMB and OPD substrates.

Features of the QuantaBlu Fluorogenic Peroxidase Substrate Kit:

• More sensitive than TMB, OPD or ABTS substrates
• Flexible - stopped, non-stopped or kinetic assays possible
• Large dynamic range (4 log peroxidase concentration range)
• Excellent stability: working solution is stable for 24 hours
• Large stokes shift; excitation/emission maxima of 325/420; range of 315-345/370-460
• Does not photobleach

A variety of substrates are available for detecting peroxidase activity in ELISA-based assays. Colorimetric substrates (e.g., TMB, OPD and ABTS) have been widely used for years. Each of these substrates varies greatly with respect to its performance characteristics such as detection sensitivity, working range and attainable signal-to-noise ratios. Substrate flexibility is also a key issue that affects an assay. Stability, development time requirements and the capacity to perform stopped and/or kinetic assays vary significantly among these substrates. Ideally, a substrate is stable, very sensitive, has high attainable signal-to-noise ratios, possesses a broad dynamic range, and allows the user to perform stopped, non-stopped and kinetic assays.

QuantaBlu Fluorogenic Peroxidase Substrate meets all the requirements of an ideal substrate for use in peroxidase detection. QuantaBlu Substrate generates a blue fluorescent product upon reaction with peroxidase. The fluorescent product does not photobleach. Fluorometric-based detection overcomes the limitations of colorimetric substrate detection, which does not allow for quantitation of greater than four optical density units. QuantaBlu Substrate allows for stopped, non-stopped and kinetic assays to be performed. Incubation times with QuantaBlu Substrate for stopped and non-stopped assays may be varied between one to 90 minutes at either room temperature or 37°C. QuantaBlu Substrate exhibits a flat baseline in assays, which facilitates low-level detection sensitivity and allows for high signal-to-noise ratios.

Related Products
QuantaBlu™ NS/K Fluorogenic Substrate Kit

OPD (o-phenylenediamine dihydrochloride) (Thermo Scientific™)

Thermo Scientific Pierce OPD is a water-soluble substrate for horseradish peroxidase (HRP) that produces a yellow-orange product detectable at 492nm by ELISA plate readers.

OPD yields a water soluble yellow-orange product when reacted with peroxidase with an absorbance maximum of 492. OPD can easily be dissolved in a substrate buffer such as Stable Peroxide Substrate Buffer (Part No. 34062).

Related Products
OPD Substrate Tablets (o-phenylenediamine dihydrochloride)

1-Step™ Slow TMB-ELISA Substrate Solution (Thermo Scientific™)

Thermo Scientific Pierce 1-Step Slow TMB ELISA Substrate Solution detects horseradish peroxidase (HRP) activity, yielding a blue color (Amax = 370nm and 652nm) that changes to yellow (Amax = 450nm) upon addition of a sulfuric or phosphoric acid stop solution. It is formulated for kinetic studies where rapid reaction is secondary to accuracy.

Features of 1-Step Slow TMB ELISA Substrate Solution :

• Available in traditional or ready-to-use formulations
• No additional reagents or filtering required
• Non carcinogenic
• Various levels of sensitivity and development speed to suit any assay
• No DMF or DMSO present in the reagents

TMB is the most popular chromogenic substrate for HRP detection in ELISA and is available in several formats. The Pierce 1-Step TMB Substrates are one-component substrates that require no preparation before use. Unlike other commercially available substrates, these products contain no DMF or DMSO.
Thermo Scientific Pierce 1-Step Ultra TMB yields the greatest sensitivity among the TMB substrates, followed by 1-Step Turbo TMB and 1-Step Slow TMB. The sensitivity of the 1-Step Turbo TMB is similar to OPD-based substrates formulated at approximately 1 mg/mL. 1-Step Slow TMB has intermediate sensitivity—more sensitive than ABTS, but less sensitive than o-phenylenediamine dihydrochloride (OPD) or 1-Step Turbo TMB. The 1-Step Slow TMB is an ideal substrate for kinetic studies.

TMB (3,3',5,5'-tetramethylbenzidine) is a chromogen that yields a blue color when oxidized, typically as a result of oxygen radicals produced by the hydrolysis of hydrogen peroxide by HRP. For kinetic or non-stopped ELISA assays, the TMB chromogen has maximal absorbances at 370nm and 652nm. The color then changes to yellow with the addition of sulfuric or phosphoric acid with maximum absorbance at 450nm. A green reaction product may result from partial conversion to the yellow product from the blue intermediate. TMB is very sensitive ELISA substrate and is more quickly oxidized than other HRP substrates, resulting in faster color development.

Related Products
1-Step™ Ultra TMB-ELISA Substrate Solution
Pierce™ TMB Substrate Kit
1-Step™ Turbo TMB-ELISA Substrate Solution

eBioscience™ DAB Advanced Chromogenic Kit (Invitrogen™)

This DAB Advanced Chromogenic Kit contains the necessary reagents to prepare a DAB substrate working solution. The enzyme peroxidase (Horseradish Peroxidase/HRP) utilizes the DAB substrate (3, 3’-diaminobenzidine tetrahydrochloride) producing a brownish-red precipitate. These precipiates are insoluble in organic solvent. This enzyme/substrate reaction can be used on tissue sections or cells (grown on culture slides or cytospins) to visualize the localization of an HRP-conjugated antibody in immunocytochemistry and immunohistochemistry applications.

Reported Application
Microscopy, Immunohistochemical Staining, Western Blotting, Immunocytochemistry

eBioscience™ TMB Solution (1X) (Invitrogen™)

The 1x eBioscience TMB Solution is a sensitive chromogenic substrate supplied as single ready-to-use solution. TMB (3,3’,5,5’-Tetramethylbenzidine) is a substrate for horseradish peroxidase (HRP) and is recommended for Ready-SET-Go! ELISA kits.

During the enzymatic reaction the TMB substrate will result in a blue color that can be read on a spectrophotometer at 650 nm. This kinetic reaction can be stopped using 1M phosphoric acid resulting in a color change to yellow that can be read at 450 nm.

Reported Application
ELISA

Chloronaphthol Substrate Powder (4-chloro-1-naphthol) (Thermo Scientific™)

Thermo Scientific Pierce Chloronaphthol (4-CN) Substrates powder is 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

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1-Step™ Chloronaphthol Substrate Solution

Amplex™ UltraRed Reagent (Invitrogen™)

Our Amplex UltraRed reagent improves upon the performance of our unique Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in peroxidase or peroxidase-coupled enzyme assays. The Amplex UltraRed reagent is also less sensitive to pH, and exhibits improved stability in the presence of hydrogen peroxide (H2O2) or thiols such as dithiothreitol (DTT).

Amplex™ Red Reagent (Invitrogen™)

The Amplex Red reagent, a highly sensitive and stable probe for H2O2, is our best fluorogenic substrate for peroxidase. In the presence of horseradish peroxidase (HRP), the Amplex Red reagent reacts in a 1:1 stoichiometry with H2O2 to produce highly fluorescent resorufin. Because H2O2 is produced in many different enzymatic reactions, the Amplex Red reagent can be used to detect the activity of many different enzymes.

Stable DAB (Invitrogen™)

Stable DAB is a stable, pre-diluted chromogen system designed to clearly localize tissue, DNA, mRNA, and antigens in in situ hybridization and immunoperoxidase procedures. Stable DAB is a ready-to-use reagent containing the correct amounts of diaminobenzidine and hydrogen peroxide. Stable DAB does not fade, creating a permanent record of results under a standard coverslip. It is color coded red for easy user identification.

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

QuantaBlu™ NS/K Fluorogenic Substrate Kit (Thermo Scientific™)

The Thermo Scientific QuantaBlu NS/K Fluorogenic Substrate Kit is a two-component kit containing proprietary fluorescent substrate and peroxide solutions for non-stopped kinetic assays. QuantaBlu Fluorogenic Peroxidase Substrates allow rapid kinetic ELISA assays to be performed with greater sensitivity than TMB and OPD substrates

Features of the QuantaBlu NS/K Fluorogenic Substrate Kit:

• More sensitive than TMB, OPD or ABTS substrates
• Large dynamic range (4 log peroxidase concentration range)
• Excellent stability: working solution is stable for 24 hours
• Large stokes shift; excitation/emission maxima of 325/420; range of 315-345/370-460
• Does not photobleach

A variety of substrates are available for detecting peroxidase activity in ELISA-based assays. Colorimetric substrates (e.g., TMB, OPD and ABTS) have been widely used for years. Each of these substrates varies greatly with respect to its performance characteristics such as detection sensitivity, working range and attainable signal-to-noise ratios.

QuantaBlu Fluorogenic Peroxidase Substrate meets all the requirements of an ideal substrate for use in peroxidase detection. QuantaBlu Substrate generates a blue fluorescent product upon reaction with peroxidase. The fluorescent product does not photobleach. Fluorometric-based detection overcomes the limitations of colorimetric substrate detection, which does not allow for quantitation of greater than four optical density units. QuantaBlu Substrate exhibits a flat baseline in assays, which facilitates low-level detection sensitivity and allows for high signal-to-noise ratios.

Related Products
QuantaBlu™ Fluorogenic Peroxidase Substrate Kit

SuperSignal™ Western Blot Substrate Bundle, Pico PLUS + trial-size Femto (Thermo Scientific™)

This substrate kit is comprised of SuperSignal West Pico PLUS Chemiluminescent Substrate plus a sample size of SuperSignal West Femto Maximum Sensitivity Substrate to provide chemiluminescent detection substrates for both routine and high sensitivity western blotting.

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 best for routine, everyday western blotting applications, providing excellent signal intensity, sensitivity and duration across a wide range of targets.

Sensitivity: pictogram to femtogram sensitivity
Stability: 8-hr working solution stability; 1-year kit stability 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)

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SuperSignal West Femto Maximum Sensitivity Substrate
Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate is an ultra-sensitive enhanced chemiluminescent (ECL) substrate for low femtogram protein level detection by western blot analysis with horseradish peroxidase (HRP) enzyme. It is optimized to detect low abundant and/or difficult to detect targets.

Sensitivity: low-femtogram (mid-zeptomole)
Stability: 8-hr working solution stability, 1-year kit stability at 4°C
Compatibility: nitrocellulose and PVDF membranes
Signal duration: up to 8 hours
Recommended primary antibody concentration: 1:5,000–1:100,000 dilution (10–200 ng/mL)
Recommended secondary antibody concentration: 1:100,000–1:500,000 dilution (2–10 ng/mL)

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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)

Novex™ ECL Chemiluminescent Substrate Reagent Kit (Invitrogen™)

Novex® ECL is a Chemiluminescent substrate used for chemiluminescence-based immunodetection of horse radish peroxidase (HRP) on western or dot blot membranes. It is a two-part reagent consisting of Reagent A (luminol) and Reagent B (an enhancer) used in equal volumes to attain the most intense light emission.