G protein coupled receptor - western blot - (Oct/11/2006 )
Im trying to look a t a membrane bound protein using weestern blot. I used NP-40 lysis buffer but i obtained very cery very faint band at the expected location. So I think that the protein didnt get solubilized.
what is the best protocol-lysis procedure i should use for membrane bound proteins.
thanks a bunch
I think your problem is in fact the detergent. NP-40 is not strong enough to disrupt the entire cell membrane in several cell types, so most or the GPCR you're looking for is lost with centrifugation. I suggest you use triton x-100 combined with other, for example SDS. Moreover maybe you'll need to increase the ionic force of your lysis buffer. For example i've used the following buffer with good results in monkey and mouse fibroblasts:
10 mM Tris-HCl pH 7.4, 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.05% SDS complemented with proteases inhibitors. Centrifuge 15 mins to 12,700 g at 4ºC (preference).
I hope it helps you.