Tetramethylbenzidine (TMB) is an excellent substrate for detecting horseradish peroxidase (HRP) labeled probes. TMB produces a soluble end product which is blue in color that is quantitated at 650nm, providing an ultrasensitive quantitative substrate system.
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 wth an adsorption maximum at 653nm. 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 = 5.9x10e4 mol-1 cm-1) provide a remarkably sensitive system for the assay of HRP and HRP labeled probes.
TMB is a single component reagent stable at room temperature and 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 usually employed in immunologic assays.
TMB reagents for ELISA and blotting techniques have been available for several years. Histochemically, TMB has been utilized primarily for the study of retrograde axonal transport. To prevent needle formation during color development, sodium nitroprusside in combination with a low pH was used.
Recent publications have described stabilization of the TMB precipitate with ammonium hexamolybdate or sodium tungstate. Methyl salicylate has also been employed to render the TMB reaction product insoluble in alcohols.
United States Biological supplies a stable, single component TMB for histochemical use containing none of these stabilizers and enhancers. The final reaction product is not affected by ethanol, xylene or xylene substitutes. Thus dehydration through alcohols to xylene and mounting the sections in a xylene type medium is possible.
TMB may also be used for blotting procedures.