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  • T0440 - 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA

T0440 Sigma-Aldrich

3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA

peroxidase substrate

Synonym: TMB

  •  NACRES NA.83



General description

TMB substrate is a chromogenic substrate for horseradish peroxidase - HRP.


TMB substrate is used in ELISA procedures, which utilize horseradish peroxidase conjugates. TMB substrate develops a soluble blue reaction product that can be read at 370 or 655 nm at 370 or 655 nm. The reaction is stopped with acid, forming a yellow reaction product which enables accurate intensity measurement at 450 nm.

How to use our TMB substrate:
• Our TMB substrate is supplied as a ready-to-use peroxidase substrate containing TMB in a mildly acidic buffer. Prior to reaction with peroxidase, the substrate should be a colorless to light bluish-green solution. The substrate system develops a blue reaction product when reacted with peroxidase.
• Upon reaching your assay endpoint with addition of acid, measure the TMB substrate intensity at 450 nm.
• Our TMB Substrate is recommended for ELISA (microwell) procedures, and has been used in various assays, as a substrate for horseradish peroxidase antibody conjugate for detecting hendra virus glycoprotein, tetracosactide, sIgA and neutrophil extracellular traps (NETs).
• 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System is not recommended for membrane applications. Since TMB substrate produces a soluble reaction product, it is not recommended for histochemistry or blotting.
Note:TMB substrate is light-sensitive and should be protected from direct sunlight or UV sources.

Safety & Documentation

Safety Information

NONH for all modes of transport
WGK Germany 
Flash Point(F) 
Not applicable
Flash Point(C) 
Not applicable

Frequently Asked Questions

Which document(s) contains shelf-life or expiration date information for a given product?
If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.
How do I get lot-specific information or a Certificate of Analysis?
The lot specific COA document can be found by entering the lot number above under the "Documents" section.
Do I need to add hydrogen peroxide to Product T0440, 3, 3´,5,5´-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA?
No hydrogen peroxide should be added to product T0440. The product, which containsa peroxidase substrate,already contains hydrogen peroxide.This solution is ready to use for ELISA assays.
What is the concentration of the TMB and what is the solvent in Product T0440, 3, 3´,5,5´-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA?
The concentration of TMB in this formulation and the identity of the solvent are considered proprietary information. The product is a ready-to-use peroxidase substrate containing TMB in a mildly acidic buffer.
Do I need to dilute Product T0440, 3, 3´,5,5´-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA?
No, the product should not be diluted.. The product is ready-to-use for ELISA applications. If the intensity of the reaction needs to be reduced, it is recommended that the antibodies orconjugates be diluted. Usage informationis described on our data sheet.
What is the difference between products T0440, 3, 3´,5,5´-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA,and T8665, 3,3´,5,5´-Tetramethylbenzidine (TMB) Liquid Substrate System?
Both products are comparable and either one can be used in ELISA procedures. The formulations which are proprietary may have some slight differences.
How do I find price and availability?
There are several ways to find pricing and availability for our products. Once you log onto our website, you will find the price and availability displayed on the product detail page. You can contact any of our Customer Sales and Service offices to receive a quote. USA customers: 1-800-325-3010 or view local office numbers.
What is the Department of Transportation shipping information for this product?
Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.
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