e-cadherin and calcium in an elisa - (May/26/2012 )
I recently did an ELISA with E-cadherin plated onto a 96-well plate then protein then primary anti and then finally secondary antibody. After I had finished it, I thought about the fact that when I plated my protein I had it in PBS with NO calcium. My e-cadherin was plated in PBS/Mg/Ca o/n and the next day it was washed in PBS-T (just normal PBS) and then incubated with my protein for an hour in PBS-T. Just wondering if anyone knows if this will affect my e-cadherin...seeing as it's calcium dependent, I read that with calcium e-cad is a rope like structure and w/o it its flimsy...I know thats in a in vivo situation but what about an in vitro one? Would adding JUST PBS undo the effects of e-cadherin that has already been plated?
It depends on the antibody you used for the ELISA, if the antibody only recognizes the native form, (which has calcium dependent folding?), then you could have a problem with detection; However, I suspect that the calcium will be well bound up in the structure, so it may not be a problem after all.
If the ELISA antibody detects the denatured form, or either native or denatured then you shouldn't have a problem at all.