Shop All Horseradish Peroxidase (HRP) Substrates

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 ›
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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.

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

SuperSignal™ ELISA Femto Substrate (Thermo Scientific™)

Thermo Scientific SuperSignal ELISA Femto Substrate is formulated for superior protein detection and low-end linearity in chemiluminescent ELISA applications.

Features of SuperSignal ELISA Femto Substrate:

Immediate light generation—intense signal generated immediately both at room temperature and 37°C; emits light at 425nm
Improved low-end linearity—easy detection of low quantities of proteins with high signal:noise ratios and low-end linearity of dose response curves
High sensitivity—femtogram-level detection of target proteins in ELISA
Reduction in assay time—high sensitivity allows for reduction in ELISA incubation steps
Stability—storage for six months at room temperature or a minimum of 12 months at 4°C with a six-hour working solution stability

SuperSignal ELISA Femto Substrate provides greater sensitivity, in ELISAs or any other solution-based assay, using chemiluminescent substrates for enzyme detection and quantification. These ELISAs can take place in either a test tube or a microplate and are quantified by measuring relative light units (RLU) in a luminometer.

SuperSignal ELISA Substrate uses an improved enhancer system that meet the needs of high-throughput screening (HTS) applications and diagnostic assays. SuperSignal ELISA Femto Substrate generates detectable light within one minute of addition to trace amounts of soluble HRP, saving up to 30 minutes per assay. This feature makes SuperSignal ELISA Femto Substrate ideal for HTS applications with the capability of running as many as 100,000 assays on robotic equipment.

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 allows researchers to detect the activity of many different enzymes (Amplex Red Assay Kits).

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

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

ABTS Substrate Tablets (Thermo Scientific™)

ABTS Substrate is a water-soluble peroxidase substrate that yields a measurable green end product for use in ELISA methods.

ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is a water-soluble HRP substrate that yields a green end product upon reaction with peroxidase. The green product has two major absorbance peaks, 410nm and 650nm. ABTS is less sensitive than OPD and TMB in ELISA applications. It is less readily oxidized, and its color development is slower (approximately 20 minutes). This may be advantageous if unacceptable background results from the use of the OPD or TMB substrates due to higher sensitivities.

Related Products
1-Step™ ABTS Substrate Solution

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

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).

SuperSignal™ ELISA Pico Chemiluminescent Substrate (Thermo Scientific™)

Thermo Scientific SuperSignal ELISA Pico Chemiluminescent Substrate is optimized to generate an intense light signal and provide exceptional performance in luminometer-based assays.

Features of SuperSignal ELISA Pico Chemiluminescent Substrate:

Immediate light generation—intense signal is produced immediately at room temperature or at 37°C; emits light at 425nm
High signal:noise ratio—minimal background
Low picogram sensitivity—detects protein in ELISAs down to the picogram levels
Room temperature storage—product with ambient shipping and room temperature storage conditions
8-hour working solution stability—consistent performance of the working solution over an 8-hour period with only a 10% decrease in activity at 24 hours
Flexible—signal can be read in black or white opaque plates

SuperSignal ELISA Pico Chemiluminescent Substrate offers greater sensitivity in ELISAs or any other solution-based assay, using chemiluminescent substrates for enzyme detection and quantification. These ELISAs can take place in either a test tube or a microplate and are quantified by measuring relative light units (RLU) in a luminometer.

Chemiluminescent substrates differ from other substrates in that the light detected is a transient product of the reaction that is only present while the enzyme-substrate reaction is occurring. This is in contrast to substrates that produce a stable, colored product; these colors persist in the well after the enzyme-substrate reaction has terminated.

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

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-ELISA Substrate Solution (Thermo Scientific™)

Thermo Scientific Pierce 1-Step Ultra TMB ELISA Substrate (250 mL) 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. 1-Step Ultra TMB is the most sensitive of the chromagenic substrates.

Features of 1-Step Ultra TMB ELISA Substrate:

• 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
Pierce™ TMB Substrate Kit
1-Step™ Turbo TMB-ELISA Substrate Solution
1-Step™ Slow TMB-ELISA Substrate Solution

SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific™)

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.

SuperSignal West Femto Maximum Sensitivity Substrate characteristics include:
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)

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

SuperSignal West Femto Maximum Sensitivity Substrate is designed for low femtogram-level detection by western blot analysis. When combined with optimized antibody concentrations and blocking buffers, SuperSignal West Femto substrate enables detection of target proteins in amounts that are too small to be seen with typical 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.