I am studying an interaction between 2 proteins (both of them have Ig domains). I am trying to prove their interaction by staining cells which express the protein A with the biotylnated ECD (extra cellular domain) of protein B through flow cytomettry.
Because it is not an antibody staining, the binding is very weak. if I just use a normal FACS buffer I can not see any binding. However, If I use a special buffer which contain 10mM CaCl2, 10mM MgCl2, 5% BSA then I can see a very strong binding in flow. But using this buffer also generated a lot of non specific binding when using a parent cell which does not express the protein A. The ECD binds to the parent cells too!
Beside that I am planing to do a co-IP experiment soon, therefore I think it is important to know the effect of these cations before setting up the Co-IP.
Hi, I also encountered similar problems, no binding of proteins. Have you solve your problem?
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