Chemiluminescence Detection on Blotting Membranes

AP as well as HRP as part of a ligand/ligate (antigen/antibody) complex is detected very sensitively by chemiluminescence. 

Substrates for AP are, e.g.,AMPPD (disodium 3-(4-methoxyspi-ro 1 2-dioxethane-3,2 -ticyclo[3.3.1.1 ] decan -4-yl)phenyl phosphate) or CSPD (disodium 3-(4-methoxyspiro l,2-dioxethane-3,2 -(5 -chloro)tricyclo [3.3.1.1] decan -4-yl)-phenyl phosphate). 

HRP splits hydroperoxides and resulting oxygen reacts with lu-minol, which gives chemiluminescence. Important The different substrates follow different kinetics, i.e., chemiluminescence of lu-minol starts very quickly and also declines soon, and AP-produced chemiluminescence raises and disappears slowly. 

After exposure to an X-ray film or photography in the dark room, the blot can be rinsed with TBS and used for a development with a chromogenic substrate. 

---