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

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Physical properties

Molecular weight

240.34

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Refrigerated (2-8 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

CAS

54827-17-7

Molecular weight

240.34

Horseradish peroxidase (HRP) and HRP conjugates facilitate the TMB oxidation in the presence of hydrogen peroxide, turning TMB into its blue 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.

Platform

Absorbance microplate reader

Absorbance	650 nm
Recommended plate	Clear bottom

Example protocol

AT A GLANCE

Important

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

SAMPLE EXPERIMENTAL PROTOCOL

Wash the assay plate following the incubation of HRP-labeled reagent.
Add 100 µL of ReadiUse™ TMB Solution into each well.
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.
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.

Calculators

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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	416.077 µL	2.08 mL	4.161 mL	20.804 mL	41.608 mL
5 mM	83.215 µL	416.077 µL	832.154 µL	4.161 mL	8.322 mL
10 mM	41.608 µL	208.039 µL	416.077 µL	2.08 mL	4.161 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Images

Figure 1. HRP dose response was measured with ReadiUse™ TMB Substrate Solution in a clear 96-well plate using a SpectraMax microplate reader (Molecular Devices).

Citations

View all 1 citations: Citation Explorer

Differential Effect of Chronic Morphine on Neuronal Degeneration in Male vs. Female Mice
Authors: Brazile Jr, Chet and Fan, Ruping and Benoit, Beau and Arnold, Thomas and Korneeva, Nadejda
Journal: Pathophysiology (2024): 152--165

References

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