Shop All Horseradish Peroxidase (HRP) Substrates

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

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)

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)

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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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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.

QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit (Thermo Scientific™)

The Thermo Scientific QuantaRed Enhanced Chemifluorescent HRP Substrate (ADHP) is the most sensitive fluorescent ELISA substrate, providing sensitivity comparable to enhanced chemiluminescence. This kit contains QuantaRed ADHP concentrate, QuantaRed Enhancer Solution, QuantaRed Stable Peroxide Solution, and QuantRed Stop Solution, and is sufficient for 960 wells (10 x 96-well plates at 100 µL working solution per well.

Features of the QuantaRed Enhanced Chemifluorescent HRP Substrate Kit:

Sensitive—low picogram to femtogram detection
Red-Shifted - minimal autofluorescence interference from biological samples
Convenient—compatible with existing filter sets, no need to purchase special filter sets
Complete—mix and use reagents, no buffers to prepare or reagents to dissolve
Flexible—use Stop Solution to effectively stop the reaction resulting in stable signal for at least 4 hours or more.
Versatile—produces a strong colorimetric signal and can be read fluorescently or colorimetrically at 570nm

QuantaRed HRP Substrate enables high-yield and sensitive fluorescent and colorimetric detection of peroxidase activity in the ELISA format. QuantaRed Substrate uses a proprietary technology to increase fluorescent yield and sensitivity of the well-documented ADHP (10-Acetyl-3,7-dihydroxyphenoxazine) chemifluorescence reaction. ADHP is a non-fluorescent compound that reacts with horseradish peroxidase (HRP) to produce resorufin, a soluble, highly fluorescent reaction product with excitation/emission maxima of ~570/585nm. The long wavelength emission of the resorufin minimizes interference from the low wavelength (blue and green) autofluorescence, making it an ideal substrate for quantitating target molecules in biological samples. In addition, the reaction product of QuantaRed Substrate is colorimetric as well as being fluorescent and can be read spectrophotmetrically at ~570nm.

Specially formulated, QuantaRed Substrate incorporates chemical enhancers for maximum sensitivity and superior low end linearity. With high quantum yield and large extinction coefficient, QuantaRed Enhanced HRP Substrate has sensitivity comparable to enhanced chemiluminescence and is more sensitive than the Amplex Red substrates for the detection of HRP in ELISA applications.

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™ 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™ Turbo TMB-ELISA Substrate Solution (Thermo Scientific™)

Thermo Scientific Pierce 1-Step Turbo TMB-ELISA Substrate Solution rapidly 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. Reactions are typically complete between five and thirty minutes after addition to HRP-Conjugates.

Features of 1-Step Turbo 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™ 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™ 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)

AEC Solution (IHC)

This Single Solution utilizes aminoethyl carbazole (AEC), an alcohol soluble chromogen, and generates a reddish deposit at the reaction site on tissue or cell preparations. Single Solution AEC is suitable for use with manual or automated staining systems, and is provided as a liquid in a single bottle that can be stored at -8°C.