alkaline phosphatase and PBS - (Apr/18/2006 )
I'm running an indirect ELISA and incubating my plate with an anti-human alkaline phosphatase. The antibody is diluted in 1% chicken egg albumin + 0.05% tween 20 made in PBS. Is it possible the PBS is rendering the AP inactive? If so, can anyone suggest an alternative buffer? Thanks!
the phosphate in pbs may inhibit the ap. you can avoid this by either performing a water wash before adding your substrate (which is not in phosphate buffer) or by using tbs (various formulations, use the one of your choice) instead of pbs.
hi Jody ! AP is readily inactivated by EDTA. it is also inactivated by inorganic orthophosphate and PBS containing disodium hydrogen orthophosphate inhibits AP activity.
try using a different buffer system for AP.
also L-phenyl alanine is a non competitive inhibitor of AP.
all the best
best thing in my opinion is to use simple saline instead of PBS. works well.