Practice question

Alkaline phosphatase is employed as a chromogenic reporter in blotting and in situ hybridization techniques. The artificial substrate 5-bromo-4-chloro-3-indolyl phosphate, BCIP, is dephosphorylated by the enzyme to an indoxyl intermediate that dimerizes oxidatively to produce insoluble indigo. The electrons released reduce nitro-blue tetrazolium, NBT, to diformazan, an insoluble dark precipitate. Co-precipitation of indigo and diformazan at the site of enzymatic activity yields an intense blue-purple color, providing sensitive localization of tagged nucleic acids or antigen-antibody complexes on membranes and tissue sections.