Shop All Horseradish Peroxidase (HRP) Substrates

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.

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 Plus Western Blotting Substrate (Thermo Scientific™)

Thermo Scientific Pierce ECL Plus Western Blotting Substrate is an acridan-based chemiluminescent and chemifluorescent horseradish peroxidase (HRP) substrate for western blot detection using X-ray film or CCD or laser-based imagers.

Pierce ECL Plus Western Blotting Substrate characteristics include:
Sensitivity: low-picogram to mid-femtogram
Stability: 1-hr working solution stability, 1-year kit stability at 4°C
Compatibility: nitrocellulose and PVDF membranes
Signal duration: up to 5 hours
Recommended primary antibody concentration: 1:1,000–1:20,000 dilution (0.05–1 µg/mL)
Recommended secondary antibody concentration: 1:25,000–1:200,000 dilution (5–40 ng/mL)
Signal: chemiluminescent and fluorescent with absorption/emission maxima of 430/503 nm

Compare and learn more about our western blot chemiluminescent substrates ›
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Pierce ECL Plus substrate generates acridinium esters when it reacts with HRP. As these ester intermediates react with peroxide, they produce a strong and sustained chemiluminescent signal that can be captured by X-ray film or CCD imaging. The substrate also produces a robust fluorescent signal that can be captured with fluorescence imagers. Pierce ECL Plus substrate enables the detection of low-picogram amounts of target protein on nitrocellulose or PVDF membrane when probed with appropriate primary and secondary antibody concentrations. Its broad dynamic range, high sensitivity and long-lasting signal make Pierce ECL Plus substrate an excellent choice for western blot analysis.

This product is sold as Pierce ECL 2 Substrate (Cat. No. PI80196) through Fisher Scientific and other channels.

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 ›
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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.

SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific™)

Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate is an enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate that enables picogram to high femtogram-level protein detection by western blot analysis.

SuperSignal West Pico PLUS Chemiluminescent Substrate characteristics include:
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)

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

SuperSignal West Pico PLUS substrate is designed to provide excellent signal intensity and sensitivity for western blotting with HRP conjugates. The intensity of the light emission combined with the exceptional duration allows for the acquisition of multiple exposures to more easily obtain publication-quality blot images. SuperSignal West Pico PLUS substrate is compatible with different membranes, blocking reagents, and a wide range of antibody dilutions, making it an ideal choice for most western blotting applications.

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)

Super AquaBlue ELISA Substrate (Invitrogen™)

Our new Super AquaBlue ELISA Substrate is an enhanced ABTS (2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) substrate solution for ELISA which provides high signal and sensitivity, low background. It is safe, stable, and extremely convenient to use. Like conventional ABTS substrate solutions, our new Super AquaBlue ELISA Substrate is oxidized by horse-radish peroxidase (HRP) - labeled probes resulting in a soluble, blue-green colored end product, whose absorption is measurable by spectrophotometer at 405 nm.

Unlike other ABTS solutions, Super AquaBlue ELISA Substrate is highly sensitive, extremely stable (18 months at room temperature or 24 months at 4°C). It is engineered for convenience as a single component (no additional H2O2 or other components are necessary).

Super AquaBlue ELISA Substrate is suitable for endpoint reactions, but is also ideal for kinetic assays; stopping the reaction with acid stop solution does not change the color or absorption properties of the blue-green end product.

Reported Application
ELISA

1-Step™ TMB-Blotting Substrate Solution (Thermo Scientific™)

Thermo Scientific Pierce TMB-Blotting 1-Step Solution is a ready-to-use solution of the blue TMB peroxidase (HRP) substrate that is specially formulated for chromogenic detection in Western blot and IHC experiments.

TMB (3,3',5,5'-tetramethylbenzidine; MW = 240.4) is most often used as a soluble substrate to detect the activity of peroxidase-conjugated antibodies in ELISA. Pierce TMB-Blotting Solution is formulated to precipitate and localize the blue reaction product onto membrane surfaces or tissue samples at sites where the peroxidase-substrate reaction occurs. TMB-Blotting Substrate produces a very large signal-to-noise ratio, although its high sensitivity requires careful washing and blocking to avoid background staining. When performing immunohistochemical techniques, aqueous counterstains and mounting medium must be used.

Features of 1-Step TMB-Blotting Solution:

HRP substrate—for sensitive detection of horseradish peroxidase activity on solid media
Chromogenic—no special equipment needed for visualization; produces dark blue bands in Western blots; is not as susceptible to fading as AEC or 4-CN substrates
Ready-to-use—no organic solvents are required to dissolve; no dilution necessary for use
Complete—does not require other buffers or addition of hydrogen peroxide

Requires:
• For best results, use TMB-Blotting Substrate with blots that have been blocked with Thermo Scientific SuperBlock Blocking Buffers for Blotting (Part No. 37517, 37537)

1-Step™ ABTS Substrate Solution (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
ABTS Substrate Tablets

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

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

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)

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 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)

View full product information ›

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.

Novex™ HRP Chromogenic Substrate (TMB) (Invitrogen™)

Novex® HRP Chromogenic Substrate is a ready to use single component reagent used for sensitive detection of a horse radish peroxidase (HRP) on western blot and dot blot membranes. Consisting of a ready-to-use solution of TMB, the Novex® HRP Chromogenic Substrate forms a blue precipatate up reaction with horse radish peroxidase (HRP).

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.

TMB Chromogen Solution (for ELISA) (Invitrogen™)

TMB (3,3', 5,5;-tetramethylbenzidine) chromogen solution may be used as a substrate for horseradish peroxidase (HRP) in ELISAs.

Using TMB
Use this reagent as supplied, no dilution or further preparation is required. In the presence of peroxidase (HRP, POD), TMB will be converted to a blue color that can be read at OD650 nm.

Pierce™ TMB Substrate Kit (Thermo Scientific™)

Thermo Scientific Pierce TMB ELISA Substrates detect 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.

Features of Thermo Scientific Pierce 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
1-Step™ Ultra TMB-ELISA Substrate Solution
1-Step™ Turbo TMB-ELISA Substrate Solution
1-Step™ Slow TMB-ELISA Substrate Solution

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

Related Products
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)

View full product information ›

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.

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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.