MilliporeSigma
All Photos(1)

Documents

ES022

Millipore

TMB/E Ultra Sensitive, Blue, Horseradish Peroxidase Substrate (soluble)

100 ml TMB/E Ultra Sensitive, Blue, Horseradish Peroxidase Substrate (soluble) for ELISA & Immunohistochemistry.

Sign Into View Organizational & Contract Pricing

Select a Size

Synonym(s):
HRP substrate, TMB
eCl@ss:
42029053
NACRES:
NB.22

Quality Level

form

liquid

manufacturer/tradename

Chemicon®

technique(s)

ELISA: suitable
immunohistochemistry: suitable

λmax

450 nm

shipped in

wet ice

Compare Similar Items

View Full Comparison

Show Differences

1 of 4

This Item
T9455WBKLSWBULS0500
technique(s)

ELISA: suitable, immunohistochemistry: suitable

technique(s)

-

technique(s)

western blot: suitable

technique(s)

western blot: suitable

shipped in

wet ice

shipped in

-

shipped in

ambient

shipped in

ambient

Quality Level

100

Quality Level

200

Quality Level

200

Quality Level

300

manufacturer/tradename

Chemicon®

manufacturer/tradename

-

manufacturer/tradename

Immobilon®

manufacturer/tradename

-

λmax

450 nm

λmax

-

λmax

-

λmax

-

General description

TMB, (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.

Application

METHOD 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.


SAMPLE PROCEDURE:


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.

Physical form

Color Code: Yellow
REAGENT 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.

MATERIALS 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.

Storage and Stability

Maintain 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.

Warm to room temperature prior to use.

REAGENT PROVIDED: Liquid solution containing TMB, 2.08 mMol L(-1) and Hydrogen Peroxide, citric acid buffer at pH 3.3. Also contains non-toxic proprietary stabilizers.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Disclaimer

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 Class Code

12 - Non Combustible Liquids

WGK

WGK 1

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable


Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

Already Own This Product?

Documents related to the products that you have purchased in the past have been gathered in the Document Library for your convenience.

Visit the Document Library

Difficulty Finding Your Product Or Lot/Batch Number?

Product numbers are combined with Pack Sizes/Quantity when displayed on the website (example: T1503-25G). Please make sure you enter ONLY the product number in the Product Number field (example: T1503).

Example:

T1503
Product Number
-
25G
Pack Size/Quantity

Additional examples:

705578-5MG-PW

PL860-CGA/SHF-1EA

MMYOMAG-74K-13

1000309185

enter as 1.000309185)

Having trouble? Feel free to contact Technical Service for assistance.

Lot and Batch Numbers can be found on a product's label following the words 'Lot' or 'Batch'.

Aldrich Products

  • For a lot number such as TO09019TO, enter it as 09019TO (without the first two letters 'TO').

  • For a lot number with a filling-code such as 05427ES-021, enter it as 05427ES (without the filling-code '-021').

  • For a lot number with a filling-code such as STBB0728K9, enter it as STBB0728 without the filling-code 'K9'.

Not Finding What You Are Looking For?

In some cases, a COA may not be available online. If your search was unable to find the COA you can request one.

Request COA

Customers Also Viewed

Slide 1 of 5

1 of 5

Our team of scientists has experience in all areas of research including Life Science, Material Science, Chemical Synthesis, Chromatography, Analytical and many others.

Contact Technical Service