ES022-500ML | TMB/E Ultra Sensitive, Blue, Horseradish Peroxidase Substrate (soluble)

500 mL  
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      Key Spec Table

      Key Applications
      Catalogue NumberES022-500ML
      Brand Family Chemicon®
      Trade Name
      • Chemicon
      DescriptionTMB/E Ultra Sensitive, Blue, 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. ES022 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, 2.08 μMol L(-1) and Hydrogen Peroxide, citric acid buffer at pH 3.3. Also contains non-toxic proprietary stabilizers.

      REAGENTS REQUIRED: Reagents Required for Stopping the Reaction, But Not Provided

      A. 0.3 Mol/L sulphuric acid for stopping the reaction and preserving blue chromogen.

      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
      Application500 ml TMB/E Ultra Sensitive, Blue, 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, ES022 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 ES022 (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 0.3 Mol/L of sulphuric acid or 0.1% sodium fluoride and read at 450 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 Size500 mL
      Transport Information
      Supplemental Information




      Safety Data Sheet (SDS) 

      Certificates of Analysis

      TitleLot Number
      ULTRASENSITIVE TMB SOLUTION A Single Component-Soluble Substrate for Kinetic and Endpoint Assays of Horseradish Peroxidase (HRP) - 2138541 2138541
      ULTRASENSITIVE TMB SOLUTION - 2025968 2025968
      ULTRASENSITIVE TMB SOLUTION - 2049194 2049194
      ULTRASENSITIVE TMB SOLUTION - 2053921 2053921
      ULTRASENSITIVE TMB SOLUTION - 2055955 2055955
      ULTRASENSITIVE TMB SOLUTION - 2557555 2557555
      ULTRASENSITIVE TMB SOLUTION -2491115 2491115
      ULTRASENSITIVE TMB SOLUTION -2623653 2623653