Shop All Horseradish Peroxidase (HRP) Substrates

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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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

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)

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

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.

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)

View full product information ›

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.

TMB Substrate Solution (Thermo Scientific™)

Addition of sulfuric acid stop solution changes the color to yellow, enabling accurate measurement of the intensity at 450nm using a spectrophotometer or plate reader. This particular formulation is used in most of the Thermo Scientific Pierce ELISA Kits where it provides consistent performance for the sandwich ELISA technique.

Features of TMB Substrate Solution:

Product Number: N301
Size: 100 mL (sufficient for about ten 96-well microplates)
Storage: Shipped on cold packs. Store at 2-8°C immediately upon receipt. Do not use product beyond the stated expiration date.
Application: ELISA: Recommended for peroxidase-based enzyme immunoassays. Upon oxidation, TMB forms a water-soluble blue reaction product that can be measured spectrophotometrically at 605nm. Upon acidification, the reaction product becomes yellow with an absorbance peak at 450nm.
Precautions: This product is sensitive to contamination from a variety of oxidizing agents. To avoid contamination and premature expiration, avoid contacting TMB solution with any potential source of contamination. Never pipette directly from the bottle. Pour out required amount onto a tube and pipette from the tube. Do not return excess TMB to primary storage container.

Procedure:
• Add 100 µL of TMB substrate to each well containing peroxidase reaction.
• Incubate at room temperature for 15 to 30 minutes.
• Add 100 µL of stop solution (0.16M sulfuric acid, N600) to each well.
• Measure absorbance at 450nm.

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

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.

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

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.

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

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.