Western for membrane bound receptor - (Jan/25/2005 )
I would like to perform a western to look at the levels of a specific receptor but I am not sure what would be the best method to extract proteins. I am afraid I will lose the receptor in my centrifugation step if I do not extract it from the membrane with a buffer more stingent than RIPA.
I would appreciate any ideas.
i ever extracted one GPCR(membrane protein) from rat brain with a very simple buffer containing 5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA. tissues were homogenized with dounce glass homogenizer on ice in the presence of PMSF. then the sample was centrifuged at 3000rpm for 10 min at 4 centigrade. collect the supernant (it's the membrane extract) and spin the membrane extract down at> 20000rpm for 30min at 4 centigrade and you will get the membrane protein extract, wash it once with TRIS. it's pure enough for WESTERN BLOT. that's what i did before.