3,3',5,5'-Tetramethylbenzidine dihydrochloride

TMBZ is a chromogenic reagent utilized for peroxidase detection. It has been developed as an alternative to benzidine. Although the TMBZ solution is colorless, it turns bluish-green (lambdamax: 655 nm) in the presence of hydrogen peroxide and peroxidase. The structure of this bluish-green complex is thought to be a radical form of two oxidized TMBZ molecules. This peroxidase substrate is used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays (ELISA). The substrate produces a soluble end product that is pale blue in color and can be read spectrophotometrically at 370 or 620-650 nm. The TMB reaction may be stopped with 2M H2SO4 (resulting in a yellow color), and read at 450nm.