TMB, High Kinetics

3,3',5,5'-Tetramethylbenzidine (TMB) has been shown to be a safe, sensitive substrate for the assay of horseradish peroxidase (HRP). Initially, in the presence of HRP and hydrogen peroxide, a one-electron oxidation product is formed. This compound, a cation free radical, is blue in color with an adsorption maximum at 653 nm. Further reaction with HRP/H₂O₂ or acidification of the radical with acid yields the diimine terminal oxidation product adsorbing light at 450 nm. The extinction coefficient of the radical (E653 nm = 3.9 x 10⁴ mol⁻¹ cm⁻¹) and diimine (E450 nm = 5.9 x 10⁴ mol⁻¹ cm⁻¹ ) provide a remarkably sensitive system for the assay of HRP and HRP labeled probes. TMBHK, available from BioVision Inc., is a highly sensitive single component reagent that is ready to use for the quantitative detection of HRP bound to a solid phase or in free solution. TMBHK is stable at room temperature for six months and is not sensitive to normal laboratory light. It is optimized with respect to TMB and hydrogen peroxide concentrations and yields a linear response with the concentrations of HRP commonly employed in immunologic assays.