Practice question

Light-emitting detection in blotting can be achieved through bioluminescence rather than colorimetric precipitation. Firefly luciferase catalyzes ATP-dependent oxidation of D-luciferin in presence of magnesium and oxygen, producing oxyluciferin, AMP, pyrophosphate, carbon dioxide, and photon emission around 560 nm. When luciferase fusion or luciferase-conjugated antibody is immobilized at antigen site, photon release is captured by cooled CCD cameras or luminometers without external excitation, minimizing autofluorescence. HRP with TMB produces blue color, alkaline phosphatase with BCIP/NBT produces purple precipitate, and beta-galactosidase with X-gal produces blue indole, none of which emit bioluminescent light.