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 use 1% NP-40 for solubilizing my membrane associated protein. 15 minutes incubation on ice with occasional shaking works or freeze-thaw techniques works fine too. Freeze the cells in -80/dry ice/ethanol bath and thaw at 42 degree centigrade. Repeat the process three times. It works for me. Hope it works for you. Good Luck!
I have used 2% DDM and 500 mM sucrose + desired buffer in my membrane protein preps. DDM is expensive but does a good job. 1% Cholate also works well and is much cheaper.
insufficient solubilization may not the only reason for poor Western detection; check also your Wb conditions; what´s the size of your porotein of interest? as receptors are often high range sized proteins, you may have a poor transfer rate; use lower acrylamid concentration or gradient for separation phase or increase blotting time