The enzyme Horseradish Peroxidase (HRP) with TMB produces:
Horseradish peroxidase is a heme-containing oxidoreductase widely conjugated to secondary antibodies for Western detection. With hydrogen peroxide as electron acceptor, HRP oxidizes chromogenic substrate 3,3',5,5'-tetramethylbenzidine. Oxidation yields a cation radical forming a blue charge-transfer complex with maximal absorbance at 652 nm soluble or as precipitate on membrane. Reaction is rapid, sensitive, and reversible upon acidification with sulfuric acid, converting blue product to yellow diamine measurable at 450 nm. Alternative substrates give distinct colors such as brown from diamino
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
