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

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10.110.1- Dose-responseData legend Generated with Quest Graph™ HRP (mU/mL) Absorbance Hover mouse to interact
HRP dose response was measured with ReadiUse™ TMB Substrate Solution in a clear 96-well plate using a SpectraMax microplate reader (Molecular Devices).
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11003 $495


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
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Overview

MW240.34
CAS #54827-17-7
Storage Refrigerated (2-8 °C)
Minimize light exposure
Category Enzyme Detection
Horseradish Peroxidase (HRP)
Related
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.




Calculators
Common stock solution preparation

Table 1. Volume of 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.



Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =
 






Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
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.

Key parameters
Instrument:Absorbance microplate reader
Absorbance:650 nm
Recommended plate:Solid white
Sample experimental protocol
  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.
Example data analysis and figures

The reading (Absorbance) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate HRP samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator

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

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





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References

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

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

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)

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

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

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

NiO nanoparticles modified with 5,10,15,20-tetrakis(4-carboxyl pheyl)-porphyrin: Promising peroxidase mimetics for HO and glucose detection
Authors: Liu Q, Yang Y, Li H, Zhu R, Shao Q, Yang S, Xu J.
Journal: Biosens Bioelectron (2014): 147

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

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

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


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