Practice question

Alkaline phosphatase paired with nitroblue tetrazolium chloride chemistry generates insoluble formazan precipitate through reduction. NBT accepts electrons, converting from yellow soluble tetrazolium to dark purple-blue diformazan that deposits at enzymatic site. Often combined with 5-bromo-4-chloro-3-indolyl phosphate which yields indigo intermediate, enhancing localization. TMB is substrate for horseradish peroxidase, not alkaline phosphatase, giving blue. ONPG is hydrolyzed by beta-galactosidase to yellow ortho-nitrophenol. Para-nitrophenyl phosphate with phosphatase produces yellow soluble product, not purple. Thus NBT deposition indicates phosphatase activity in blotting and in situ assays.