3,3',5,5'-Tetramethylbenzidine
3,3',5,5'-Tetramethylbenzidine[edit | edit source]
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[edit | edit source]
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[edit | edit source]
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.
Applications[edit | edit source]
Enzyme-Linked Immunosorbent Assay (ELISA)[edit | edit source]
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[edit | edit source]
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[edit | edit source]
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[edit | edit source]
Gallery[edit | edit source]
Chemical structure of TMB
Oxidation of TMB
3,3',5,5'-Tetramethylbenzidine[edit | edit source]
3,3',5,5'-Tetramethylbenzidine
3,3',5,5'-Tetramethylbenzidine oxidation
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Contributors: Prab R. Tumpati, MD
