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ReadiUse™ TMB Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*

HRP dose response was measured with ReadiUse™ TMB Substrate Solution in a clear 96-well plate using a SpectraMax microplate reader (Molecular Devices).
HRP dose response was measured with ReadiUse™ TMB Substrate Solution in a clear 96-well plate using a SpectraMax microplate reader (Molecular Devices).
Ordering information
Price ()
Catalog Number11003
Unit Size
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Additional ordering information
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight240.34
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageRefrigerated (2-8 °C); Minimize light exposure


Molecular weight
Horseradish peroxidase (HRP) and HRP conjugates facilitate the ABTS oxidation in the presence of hydrogen peroxide, turning ABTS into its blue-green oxidized product. ReadiUse™ TMB Substrate Solution is a premixed solution of TMB substrate with hydrogen peroxide. It produces a blue product upon interaction with HRP or HRP conjugates without the addition of hydrogen peroxide. The soluble blue product can be quantitated at 650 nm. Use of a stop solution enhances sensitivity 2-4 fold and the resulting yellow solution can be read at 450 nm. ReadiUse™ TMB Substrate Solution provides an convenient and ultrasensitive quantitative substrate system.


Absorbance microplate reader

Recommended plateClear bottom

Example protocol


Warm ReadiUse™ TMB Solution to room temperature before use.
Note      The reagent is to be used as supplied, no dilution is required.


  1. Wash the assay plate following the incubation of HRP-labeled reagent.
  2. Add 100 µL of ReadiUse™ TMB Solution into each well.
  3. Incubate the plate at room temperature for 15 – 30 min or until the desired color develops. Note: The incubation time varies depending on the assay conditions.
  4. Measure the absorbance signal at 650 nm with an ELISA microplate reader. Note: If desired, the reaction can be stopped by adding an equal volume of 2M sulfuric acid to each well. Stopped reaction should be read at 450 nm. 


Common stock solution preparation

Table 1. Volume of appropriate solvent needed to reconstitute specific mass of ReadiUse™ TMB Substrate Solution *Optimized for ELISA Assays with HRP Conjugates* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM416.077 µL2.08 mL4.161 mL20.804 mL41.608 mL
5 mM83.215 µL416.077 µL832.154 µL4.161 mL8.322 mL
10 mM41.608 µL208.039 µL416.077 µL2.08 mL4.161 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles


View all 42 references: Citation Explorer
Peroxidase-like activity of apoferritin paired gold clusters for glucose detection
Authors: Jiang X, Sun C, Guo Y, Nie G, Xu L.
Journal: Biosens Bioelectron (2014): 165
Novel tungsten carbide nanorods: an intrinsic peroxidase mimetic with high activity and stability in aqueous and organic solvents
Authors: Li N, Yan Y, Xia BY, Wang JY, Wang X.
Journal: Biosens Bioelectron (2014): 521
Glucose-sensitive colorimetric sensor based on peroxidase mimics activity of porphyrin-Fe3O4 nanocomposites
Authors: Liu Q, Li H, Zhao Q, Zhu R, Yang Y, Jia Q, Bian B, Zhuo L.
Journal: Mater Sci Eng C Mater Biol Appl (2014): 142
Peroxidase-Like Catalytic Activity of Ag3PO4 Nanocrystals Prepared by a Colloidal Route
Authors: Liu Y, Zhu G, Yang J, Yuan A, Shen X.
Journal: PLoS One (2014): e109158
Colorimetric estimation of human glucose level using gamma-Fe(2)O(3) nanoparticles: an easily recoverable effective mimic peroxidase
Authors: Mitra K, Ghosh AB, Sarkar A, Saha N, Dutta AK.
Journal: Biochem Biophys Res Commun (2014): 30
Visual detection of blood glucose based on peroxidase-like activity of WS2 nanosheets
Authors: Lin T, Zhong L, Song Z, Guo L, Wu H, Guo Q, Chen Y, Fu F, Chen G.
Journal: Biosens Bioelectron (2014): 302
5,10,15,20-tetrakis(4-carboxyl phenyl)porphyrin-CdS nanocomposites with intrinsic peroxidase-like activity for glucose colorimetric detection
Authors: Liu Q, Jia Q, Zhu R, Shao Q, Wang D, Cui P, Ge J.
Journal: Mater Sci Eng C Mater Biol Appl (2014): 177
Biogenic magnetic nanoparticles from Burkholderia sp. YN01 exhibiting intrinsic peroxidase-like activity and their applications
Authors: Pan Y, Li N, Mu J, Zhou R, Xu Y, Cui D, Wang Y, Zhao M.
Journal: Appl Microbiol Biotechnol. (2014)
Interactions of macromolecular crowding agents and cosolutes with small-molecule substrates: effect on horseradish peroxidase activity with two different substrates
Authors: Aumiller WM, Jr., Davis BW, Hatzakis E, Keating CD.
Journal: J Phys Chem B (2014): 10624
Au nanoparticles on citrate-functionalized graphene nanosheets with a high peroxidase-like performance
Authors: Chen X, Tian X, Su B, Huang Z, Oyama M.
Journal: Dalton Trans (2014): 7449