e-cadherin and calcium in an elisa - (May/26/2012 )

Hi there,

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?

Thanks!
b_06er

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.