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 ›
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

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.

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

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

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 ›
View all HRP substrates available ›

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.

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)

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

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.

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

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.

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)

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

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).

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)

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.