3,3',5,5'-Tetramethylbenzidine

Chemical structure of 3,3',5,5'-Tetramethylbenzidine

3,3',5,5'-Tetramethylbenzidine (TMB) is an organic compound that is widely used as a chromogenic substrate in biochemistry and molecular biology applications. It is particularly known for its use in enzyme-linked immunosorbent assay (ELISA) and other immunoassay techniques.

Chemical Properties

TMB is a derivative of benzidine, with four methyl groups attached to the benzene rings. Its chemical formula is C₁₆H₂₀N₂. The presence of the methyl groups makes TMB less toxic than benzidine, which is a known carcinogen.

Mechanism of Action

TMB acts as a hydrogen donor in the presence of peroxidase enzymes, such as horseradish peroxidase (HRP). When oxidized by HRP, TMB produces a blue-colored product. This reaction is often used to detect the presence of antibodies or antigens in a sample.

Oxidation of TMB to its blue-colored product

Applications

Enzyme-Linked Immunosorbent Assay (ELISA)

In ELISA, TMB is used as a substrate for HRP-conjugated antibodies. The intensity of the blue color produced is proportional to the amount of enzyme present, which in turn is proportional to the amount of target antigen or antibody in the sample. The reaction can be stopped by adding an acid, which turns the solution yellow, allowing for quantification by measuring absorbance at 450 nm.

Other Uses

TMB is also used in Western blotting and immunohistochemistry as a chromogenic substrate. It provides a sensitive and reliable method for detecting proteins and other biomolecules.

Safety and Handling

While TMB is less hazardous than benzidine, it should still be handled with care. Appropriate laboratory safety procedures should be followed, including the use of personal protective equipment such as gloves and goggles.

Related Pages

• Enzyme-linked immunosorbent assay
• Horseradish peroxidase
• Chromogenic substrate
• Immunoassay