A part of MilliporeSigma

ES001-100ML | TMB/E Single Reagent, Blue color, Horseradish Peroxidase Substrate (soluble)

100 mL  
Retrieving price...
Price could not be retrieved
Minimum Quantity needs to be mulitiple of
Upon Order Completion More Information
You Saved ()
Request Pricing
Limited AvailabilityLimited Availability
Limited Quantities Available
    Remaining : Will advise
      Remaining : Will advise
      Will advise
      Contact Customer Service


      Contact Customer Service

      Click To Print This Page


      Replacement Information

      Key Specifications Table

      Key Applications
      Catalogue NumberES001-100ML
      Brand Family Chemicon®
      Trade Name
      • Chemicon
      DescriptionTMB/E Single Reagent, Blue color, Horseradish Peroxidase Substrate (soluble)
      OverviewTMB, (3,3',5,5'-tetramethybenzidine) has been shown to be a safe-sensitive substrate for the assay of HRP. Initially, in the presence of HRP and hydrogen peroxide, a one-electron oxidation product is formed. This compound, a cation free radical, is blue in color with an adsorption maximum of 653 nm. Further reaction with HRP/H2O2 or acidification of the radical with acid yields the diimine terminal oxidation product adsorbing light at 450 nm. The extinction coefficient of the radical (E653nm=3.9 x 104 mol-1 cm-1) and diimine (E450nm=5.9 x 104 mol-1 cm-1) provide a remarkably sensitive system for the assay of HRP and HRP labeled probes. ES001 is a single component reagent stable at room temperature and not sensitive to normal laboratory light. It is optimized with respect to TMB and hydrogen peroxide concentrations and yields a linear response with the concentrations of HRP usually employed in immunological assays.
      Product Information
      PresentationREAGENT PROVIDED:
      Liquid solution containing TMB, 1.25 mMol/L and Hydrogen Peroxide, 2.21 mMol/L and less than 1% dimethyl sulfoxide in a 0.08 Mol/L) acetate buffer at pH 4.9. Also contains non-toxic proprietary stabilizers.

      Materials Required:
      Reagents Required for Stopping the Reaction, But Not Provided
      A. Sodium fluoride, 0.1% for stopping the reaction and preserving blue chromogen. Prepare by dissolving 1 g of sodium fluoride in 1 liter of reagent grade water. Observe all precautions on label for use of sodium fluoride.

      B. Acid for stopping the reaction and converting the blue radical to the yellow diimine. Either 0.5 Mol/L sulfuric acid or 0.25 Mol/L hydrochloric acid may be used. THE ABSORBANCE MUST BE OBTAINED WITHIN 5 MINUTES AFTER ADDING THE ACID.
      Note: Reagent grade water must contain less than 10-7 Mol/L of iron or copper salts otherwise unreacted TMB will be converted non-enzymatically to the diimine.
      Color CodeYellow
      Application100 ml TMB/E Single Reagent, Blue color, Horseradish Peroxidase Substrate (soluble) for ELISA.
      Key Applications
      • ELISA
      Application NotesMETHOD SYNOPSIS:

      After completion of analyte binding to a solid phase and reaction with a HRP labeled probe, ES001 is added. Oxidation of TMB produces a blue reaction product that is measured at 650 nm. The color formation as a function of time can be recorded or the reaction stopped with sodium fluoride after a fixed interval. Increased sensitivity can be achieved by converting the blue radical to the diimine by addition of acid. The resulting yellow color is measured immediately at 450 nm.


      1. Complete all required incubations with antibodies, probes, HRP labeled reagents.

      2. Wash plate at least 4 times with PBS or Tris buffered saline containing 0.1% Tween-20.

      3. After the final wash, shake and blot all residual buffer from plate wells.

      4. Add 0.1 mL of ES001 (TMBE Solution) to appropriate wells and incubate 5-30 minutes.

      Note: The reaction time will depend upon the activity of the HRP probe. If color develops too briskly, zero order kinetics will not prevail. Dilution of the probe, antibody or HRP labeled reagent may be required.

      5. The reaction can be monitored as a function of time for kinetic assays or stopped with 0.1 mL of 0.1% sodium fluoride and read at 650 nm.

      6. If the procedure demands conversion to the yellow diimine, add 0.1 mL of either acid described in the reagent section above and record the absorbance within 5 minutes.

      Variations of time, reagent volume and temperature require standardization by the user.
      Biological Information
      Physicochemical Information
      Quantitation Wavelength450 nm
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage ConditionsMaintain at room temperature for up to six months. For long term storage, up to twelve months, maintain at 2-8°C. Protect from exposure to direct sunlight. Discard if solution is blue or turbid.
      Packaging Information
      Material Size100 mL
      Transport Information
      Supplemental Information




      Safety Data Sheet (SDS) 

      Certificates of Analysis

      TitleLot Number
      TMB/E SOLUTION 2470345
      TMB/E SOLUTION A Single Component-Soluble Substrate for Kinetic and Endpoint Assays of Horseradish Peroxidase (HRP) - 2115532 2115532
      TMB/E SOLUTION - 2556265 2556265
      TMB/E SOLUTION -2709154 2709154
      TMB/E SOLUTION -2742745 2742745
      TMB/E SOLUTION -2791185 2791185
      TMB/E SOLUTION -2791190 2791190
      TMB/E SOLUTION -2791191 2791191
      TMB/E SOLUTION A Single Component-Soluble Substrate for Kinetic and Endpoint Assays of Horseradish Peroxidase (HRP) - 2038626 2038626
      TMB/E SOLUTION A Single Component-Soluble Substrate for Kinetic and Endpoint Assays of Horseradish Peroxidase (HRP) - 2089397 2089397
      TMB/E SOLUTION A Single Component-Soluble Substrate for Kinetic and Endpoint Assays of Horseradish Peroxidase (HRP) - 2147011 2147011