Formula C16H20N2 | Molar mass 240.3482 g/mol | |
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3,3’,5,5’-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays (ELISA). TMB is a white crystal powder that forms a pale blue-green liquid in solution with ethyl acetate. TMB is degraded by sunlight and by fluorescent lights.
Contents
Enzymatic assay
TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase.
The resulting diimine causes the solution to take on a blue colour, and this colour change can be read on a spectrophotometer at a wavelength of 650 nm.
The reaction can be halted by addition of acid or another stop reagent. Using sulfuric acid turns TMB yellow. The colour may be read at 450 nm.
Material Safety
TMB should be kept out of direct sunlight as it is photosensitive. It is not known if TMB is carcinogenic and the evidence is contradictory: TMB is not mutagenic by the Ames test, and did not induce formation of tumors in a single-arm study of 24 rats. On that evidence, it has been used as a replacement for carcinogenic compounds such as benzidine and o-phenylenediamine.