ELISA plate detecting HRP with TMB substrate (blue) and color changing to yellow upon addition of sulfuric acid

TMB substrates and other reagents

Find HRP and other enzyme substrates for ELISA applications in a range of sensitivities for different methods including colorimetric, chemiluminescent, and fluorescent detection.

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Colorimetric substrates for ELISA applications

Find a variety of colorimetric (chromogenic) substrates for ELISA applications for horseradish peroxidase (HRP) and for alkaline phosphatase (AP) to meet your sensitivity and experimental needs.

Substrate Enzyme Benefits, format Catalog No. (total assays¹) Absorbance and color Detection limit² Recommended primary (1°) and secondary (2°) antibody dilutions²
TMB (3,3',5,5'-tetramethylbenzidine)
1-Step Ultra TMB HRP Most sensitive of the chromogenic substrates, ready-to-use format 34028
(2500 wells)
450 nm (650 nm) Yellow (Blue) ~2 pg/well
(20 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
1-Step Slow TMB HRP Ideal substrate for kinetic studies, ready-to-use format 34024
(2500 wells)
450 nm (652 nm)
Yellow (Blue)
~8 pg/well
(80 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
1-Step Turbo TMB HRP Rapidly detects HRP activity within 5–30 min, ready-to-use format 34022
(2500 wells)
450 nm (652 nm)
Yellow (Blue)
~7 pg/well
(70 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
TMB Substrate Kit HRP 2-part kit 34021
(4000 wells)
450 nm (652 nm)
Yellow (Blue)
~6 pg/well
(60 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
TMB Chromogen Solution HRP Ready-to-use format 00-2023 (500 mL) 450 nm (652 nm)
Yellow (Blue)
   
ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt
1-Step ABTS Substrate Solution HRP Ready-to-use format 37615
(2000 wells)
410 nm (650 nm)
Green
~250 pg/well
(2.5 ng/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
OPD (o-phenylenediamine dihydrochloride)
OPD Substrate HRP Pre-weighted tablets 34006 (tablets)
34005 (25 g)
490 nm (450 nm)
Green (Orange)
~7 pg/well
(70 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
PNPP (p-Nitrophenyl Phosphate)
1-Step PNPP Substrate Solution AP Ready-to-use format 37621
(1000 wells)
405 nm
Yellow
~10 ng/well
(100 ng/mL)
1°- 1:500
2°- 1:5,000 to 1:20,000
PNPP Substrate AP Pre-weighted tablets 37620
(105 Tablet, buffer kit)
34047
(105 Tablets)
34045
(25 g)
405 nm
Yellow
~10 ng/well
(100 ng/mL)
1°- 1:500
2°- 1:5,000 to 1:20,000
ONPG (o-nitrophenyl-β-D-galactopyranoside)
ONPG Substrate Gal   34055 (5 g) 410 nm
Yellow
~10 ng/well (100 ng/mL) 1°- 1:500
2°- 1:5,000

¹ See product instructions for additional information and assay considerations that determine the number of assays.
² Detection limits and recommended antibody dilutions (based on 1 mg/mL stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested.


Colorimetric ELISA substrates at a glance

TMB (3,3',5,5'-tetramethylbenzidine) soluble substrates yield a blue color when detecting HRP. The major absorbance maxima or the reaction product are 370 nm and 652 nm. The color then changes to yellow with the addition of sulfuric or phosphoric acid (stop solution) with maximum absorbance at 450 nm. TMB is very sensitive and may produce significant background signal if too much protein or antibody is used. TMB is more quickly oxidized than other HRP substrates, resulting in faster color development.

ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is used to detect HRP and yields a water-soluble green end reaction product. The green product has two major absorbance peaks—410 nm and 650 nm. ABTS is less sensitive than the OPD and TMB substrates for HRP detection. Color development is slow (approximately 20 minutes) which may be advantageous if unacceptable background results from the use of the OPD or TMB substrates due to higher sensitivities. ABTS is available in either tablet or a ready-to-use for formulation.

OPD (o-phenylenediamine dihydrochloride) is used to detect HRP and yields a water soluble yellow-orange reaction product. The reaction product has an absorbance maximum of 492 nm. OPD is available in either powder or tablet forms and easily prepared by dissolving in Stable Peroxide Substrate Buffer or buffered hydrogen peroxide solution.

PNPP (p-Nitrophenyl Phosphate, Disodium Salt) is a widely used substrate for detecting alkaline phosphatase in ELISA applications. PNPP produces a yellow water-soluble reaction product that absorbs light at 405 nm. PNPP is available either as a crystalline powder, 5 mg tablets, or as a ready-to-use formulation.


Chemiluminescent ELISA substrates

We offer several chemiluminescent substrates for ELISA development with horseradish peroxidase enzyme (HRP) and alkaline phosphatase (AP):

Enzyme Substrate Catalog No.
(Total Assays¹)
Emax³ Color Detection Limit² Primary (1°) and Secondary (2°) Antibody Dilutions²
HRP SuperSignal ELISA Pico 37070 (1000 wells)
37069 (2500 wells)
425 nm
Blue/Green
~500 fg/well
(5 pg/mL)
1° 1:1,000
2° 1:10,000 to 1:50,000
HRP SuperSignal ELISA Femto 37075 (1000 wells)
37074 (2500 wells)
425 nm
Blue/Green
~170 fg/well
(1.7 pg/mL)
1° 1:1,000
2° 1:50,000 to 1:100,000
AP CDP-Star Substrate T2145 (50 mL)
T2147 (250 mL)
475 nm    
AP CSPD Substrate T2141 (50 mL)
T2143 (250 mL)
475 nm    
AP DynaLight Substrate with RapidGlow Enhancer 4475406 (100 mL)
4475410 (1L)
  ~1 pg/mL  

¹ Total number of assays based on 96-well microplate. See product instructions for additional information and assay considerations that determine the number of assays.
² Detection limits and recommended antibody dilutions (based on 1 mg/mL stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.
³ The peak emission wavelength is given for reference. However, for best sensitivity, measure total light output using a luminometer.


Chemiluminescent ELISA substrates at a glance

CSPD and CDP-Star are chemiluminescent 1,2-dioxetane alkaline phosphatase substrates that emit light with a maximum light intensity at a wavelength of 475 nm. These substrates are “glow” substrates and provide a sustained maximum signal over time only after 15–60 minutes depending on temperature. They are supplied at 5 or 25 mM (respectively) in aqueous buffer. Sensitivity can be improved with addition of Sapphire-II or Emerald-II luminescence enhancers.

DynaLight Substrate with RapidGlow Enhancer is a ready-to-use chemiluminescent substrate formulation that has been optimized to achieve faster results in solution-based assays. This substrate is classified as a flash and glow substrate that provides fast and sustained maximum signal as early as 2–10 minutes. The DynaLight Substrate with RapidGlow Enhancer formulation includes 1,2-dioxetane chemiluminescent substrate and a polymeric enhancer that enables ultra-sensitive immunoassay detection by alkaline phosphatase label.

SuperSignal ELISA Pico Chemiluminescent Substrate provides excellent performance for a large range of target protein amounts and is easily optimized to detect with greater sensitivity than entry-level colorimetric substrates. Rapid signal generation with 5–30 minute signal stability depending on HRP concentration.

SuperSignal ELISA Femto Maximum Sensitivity Substrate is one of the most sensitive substrates available for ELISA applications. When properly optimized, the lower detection limit is 1 to 10 orders of magnitude lower than commonly used colorimetric substrates. However, without proper optimization, it is easy to overwhelm the system with protein and enzyme, resulting in high background and possibly negative results.


Fluorescent ELISA substrates

We offer several chemifluorescent substrates for ELISA applications with horseradish peroxidase enzyme (HRP):

Enzyme Substrate Catalog No. (Total Assays¹) Emax / Amax (Emission/Excitation) Detection Limit² Primary (1°) and Secondary (2°) Antibody Dilutions²
HRP QuantaBlu Fluorogenic Substrate 15169 (2700 wells) 420 nm/
325 nm
~500 fg/well
(5 pg/mL)
1° 1:500
2° 1:5,000 to 1:20,000
HRP QuantaBlu Kinetic Fluorogenic Substrate 15162 (2700 wells) 420 nm/
325 nm
~500 fg/well
(5 pg/mL)
1° 1:500
2° 1:5,000 to 1:20,000
HRP QuantaRed Enhanced Chemifluorescent Substrate 15159 (1000 wells) 585 nm/
570 nm
~400 fg/well
(4 pg/mL)
1° 1:1,000
2° 1:5,000 to 1:20,000
HRP Amplex Red Reagent A12222 (200 assays) 571 nm/
585 nm
   
HRP Amplex UltraRed Reagent A36006 (3400 rxns) 490–550 nm/
580–590 nm
   

¹ See product instructions for additional information and assay considerations that determine the number of assays.
² Detection limits and recommended antibody dilutions (based on 1 mg/ml stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.


Fluorescent ELISA substrates at a glance

QuantaBlu Fluorogenic Substrate has a large linear detection range with low-end linearity for detection of HRP. The stable fluorescent reaction product has an Emax/Amax of 420 nm/325 nm allowing stopped, non-stopped, and kinetic assays to be performed; an advantage over the more sensitive chemiluminescent substrates.

QuantaRed Enhanced Chemifluorescent Substrate is the most sensitive fluorescent ELISA substrate available for HRP detection. The fluorescent reaction product (resorufin) is stable for 4 hours with an Emax/Amax of 585 nm/570 nm when the reaction is stopped. The red-shifted resorufin reaction product permits detection at a wavelength with less interference from autofluorescence that can occur in biological samples.

Amplex Red Reagent is a highly sensitive and stable probe for H2O2. Because H2O2 is produced in many different enzymatic reactions, the Amplex Red reagent allows for detection of many different enzymes (Amplex Red Assay Kits).

Amplex UltraRed Reagent improves upon the performance of Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in peroxidase or peroxidase-coupled enzyme assays.
 


Colorimetric substrates for ELISA applications

Find a variety of colorimetric (chromogenic) substrates for ELISA applications for horseradish peroxidase (HRP) and for alkaline phosphatase (AP) to meet your sensitivity and experimental needs.

Substrate Enzyme Benefits, format Catalog No. (total assays¹) Absorbance and color Detection limit² Recommended primary (1°) and secondary (2°) antibody dilutions²
TMB (3,3',5,5'-tetramethylbenzidine)
1-Step Ultra TMB HRP Most sensitive of the chromogenic substrates, ready-to-use format 34028
(2500 wells)
450 nm (650 nm) Yellow (Blue) ~2 pg/well
(20 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
1-Step Slow TMB HRP Ideal substrate for kinetic studies, ready-to-use format 34024
(2500 wells)
450 nm (652 nm)
Yellow (Blue)
~8 pg/well
(80 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
1-Step Turbo TMB HRP Rapidly detects HRP activity within 5–30 min, ready-to-use format 34022
(2500 wells)
450 nm (652 nm)
Yellow (Blue)
~7 pg/well
(70 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
TMB Substrate Kit HRP 2-part kit 34021
(4000 wells)
450 nm (652 nm)
Yellow (Blue)
~6 pg/well
(60 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
TMB Chromogen Solution HRP Ready-to-use format 00-2023 (500 mL) 450 nm (652 nm)
Yellow (Blue)
   
ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt
1-Step ABTS Substrate Solution HRP Ready-to-use format 37615
(2000 wells)
410 nm (650 nm)
Green
~250 pg/well
(2.5 ng/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
OPD (o-phenylenediamine dihydrochloride)
OPD Substrate HRP Pre-weighted tablets 34006 (tablets)
34005 (25 g)
490 nm (450 nm)
Green (Orange)
~7 pg/well
(70 pg/mL)
1°- 1:1,000
2°- 1:5,000 to 1:50,000
PNPP (p-Nitrophenyl Phosphate)
1-Step PNPP Substrate Solution AP Ready-to-use format 37621
(1000 wells)
405 nm
Yellow
~10 ng/well
(100 ng/mL)
1°- 1:500
2°- 1:5,000 to 1:20,000
PNPP Substrate AP Pre-weighted tablets 37620
(105 Tablet, buffer kit)
34047
(105 Tablets)
34045
(25 g)
405 nm
Yellow
~10 ng/well
(100 ng/mL)
1°- 1:500
2°- 1:5,000 to 1:20,000
ONPG (o-nitrophenyl-β-D-galactopyranoside)
ONPG Substrate Gal   34055 (5 g) 410 nm
Yellow
~10 ng/well (100 ng/mL) 1°- 1:500
2°- 1:5,000

¹ See product instructions for additional information and assay considerations that determine the number of assays.
² Detection limits and recommended antibody dilutions (based on 1 mg/mL stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested.


Colorimetric ELISA substrates at a glance

TMB (3,3',5,5'-tetramethylbenzidine) soluble substrates yield a blue color when detecting HRP. The major absorbance maxima or the reaction product are 370 nm and 652 nm. The color then changes to yellow with the addition of sulfuric or phosphoric acid (stop solution) with maximum absorbance at 450 nm. TMB is very sensitive and may produce significant background signal if too much protein or antibody is used. TMB is more quickly oxidized than other HRP substrates, resulting in faster color development.

ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is used to detect HRP and yields a water-soluble green end reaction product. The green product has two major absorbance peaks—410 nm and 650 nm. ABTS is less sensitive than the OPD and TMB substrates for HRP detection. Color development is slow (approximately 20 minutes) which may be advantageous if unacceptable background results from the use of the OPD or TMB substrates due to higher sensitivities. ABTS is available in either tablet or a ready-to-use for formulation.

OPD (o-phenylenediamine dihydrochloride) is used to detect HRP and yields a water soluble yellow-orange reaction product. The reaction product has an absorbance maximum of 492 nm. OPD is available in either powder or tablet forms and easily prepared by dissolving in Stable Peroxide Substrate Buffer or buffered hydrogen peroxide solution.

PNPP (p-Nitrophenyl Phosphate, Disodium Salt) is a widely used substrate for detecting alkaline phosphatase in ELISA applications. PNPP produces a yellow water-soluble reaction product that absorbs light at 405 nm. PNPP is available either as a crystalline powder, 5 mg tablets, or as a ready-to-use formulation.


Chemiluminescent ELISA substrates

We offer several chemiluminescent substrates for ELISA development with horseradish peroxidase enzyme (HRP) and alkaline phosphatase (AP):

Enzyme Substrate Catalog No.
(Total Assays¹)
Emax³ Color Detection Limit² Primary (1°) and Secondary (2°) Antibody Dilutions²
HRP SuperSignal ELISA Pico 37070 (1000 wells)
37069 (2500 wells)
425 nm
Blue/Green
~500 fg/well
(5 pg/mL)
1° 1:1,000
2° 1:10,000 to 1:50,000
HRP SuperSignal ELISA Femto 37075 (1000 wells)
37074 (2500 wells)
425 nm
Blue/Green
~170 fg/well
(1.7 pg/mL)
1° 1:1,000
2° 1:50,000 to 1:100,000
AP CDP-Star Substrate T2145 (50 mL)
T2147 (250 mL)
475 nm    
AP CSPD Substrate T2141 (50 mL)
T2143 (250 mL)
475 nm    
AP DynaLight Substrate with RapidGlow Enhancer 4475406 (100 mL)
4475410 (1L)
  ~1 pg/mL  

¹ Total number of assays based on 96-well microplate. See product instructions for additional information and assay considerations that determine the number of assays.
² Detection limits and recommended antibody dilutions (based on 1 mg/mL stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.
³ The peak emission wavelength is given for reference. However, for best sensitivity, measure total light output using a luminometer.


Chemiluminescent ELISA substrates at a glance

CSPD and CDP-Star are chemiluminescent 1,2-dioxetane alkaline phosphatase substrates that emit light with a maximum light intensity at a wavelength of 475 nm. These substrates are “glow” substrates and provide a sustained maximum signal over time only after 15–60 minutes depending on temperature. They are supplied at 5 or 25 mM (respectively) in aqueous buffer. Sensitivity can be improved with addition of Sapphire-II or Emerald-II luminescence enhancers.

DynaLight Substrate with RapidGlow Enhancer is a ready-to-use chemiluminescent substrate formulation that has been optimized to achieve faster results in solution-based assays. This substrate is classified as a flash and glow substrate that provides fast and sustained maximum signal as early as 2–10 minutes. The DynaLight Substrate with RapidGlow Enhancer formulation includes 1,2-dioxetane chemiluminescent substrate and a polymeric enhancer that enables ultra-sensitive immunoassay detection by alkaline phosphatase label.

SuperSignal ELISA Pico Chemiluminescent Substrate provides excellent performance for a large range of target protein amounts and is easily optimized to detect with greater sensitivity than entry-level colorimetric substrates. Rapid signal generation with 5–30 minute signal stability depending on HRP concentration.

SuperSignal ELISA Femto Maximum Sensitivity Substrate is one of the most sensitive substrates available for ELISA applications. When properly optimized, the lower detection limit is 1 to 10 orders of magnitude lower than commonly used colorimetric substrates. However, without proper optimization, it is easy to overwhelm the system with protein and enzyme, resulting in high background and possibly negative results.


Fluorescent ELISA substrates

We offer several chemifluorescent substrates for ELISA applications with horseradish peroxidase enzyme (HRP):

Enzyme Substrate Catalog No. (Total Assays¹) Emax / Amax (Emission/Excitation) Detection Limit² Primary (1°) and Secondary (2°) Antibody Dilutions²
HRP QuantaBlu Fluorogenic Substrate 15169 (2700 wells) 420 nm/
325 nm
~500 fg/well
(5 pg/mL)
1° 1:500
2° 1:5,000 to 1:20,000
HRP QuantaBlu Kinetic Fluorogenic Substrate 15162 (2700 wells) 420 nm/
325 nm
~500 fg/well
(5 pg/mL)
1° 1:500
2° 1:5,000 to 1:20,000
HRP QuantaRed Enhanced Chemifluorescent Substrate 15159 (1000 wells) 585 nm/
570 nm
~400 fg/well
(4 pg/mL)
1° 1:1,000
2° 1:5,000 to 1:20,000
HRP Amplex Red Reagent A12222 (200 assays) 571 nm/
585 nm
   
HRP Amplex UltraRed Reagent A36006 (3400 rxns) 490–550 nm/
580–590 nm
   

¹ See product instructions for additional information and assay considerations that determine the number of assays.
² Detection limits and recommended antibody dilutions (based on 1 mg/ml stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.


Fluorescent ELISA substrates at a glance

QuantaBlu Fluorogenic Substrate has a large linear detection range with low-end linearity for detection of HRP. The stable fluorescent reaction product has an Emax/Amax of 420 nm/325 nm allowing stopped, non-stopped, and kinetic assays to be performed; an advantage over the more sensitive chemiluminescent substrates.

QuantaRed Enhanced Chemifluorescent Substrate is the most sensitive fluorescent ELISA substrate available for HRP detection. The fluorescent reaction product (resorufin) is stable for 4 hours with an Emax/Amax of 585 nm/570 nm when the reaction is stopped. The red-shifted resorufin reaction product permits detection at a wavelength with less interference from autofluorescence that can occur in biological samples.

Amplex Red Reagent is a highly sensitive and stable probe for H2O2. Because H2O2 is produced in many different enzymatic reactions, the Amplex Red reagent allows for detection of many different enzymes (Amplex Red Assay Kits).

Amplex UltraRed Reagent improves upon the performance of Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in peroxidase or peroxidase-coupled enzyme assays.
 


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