Hi All,
Help me please. I need to do western blot of GPCR. I tried several times but I donot get any band. I think my GPCR which is in CHO-K1 cells is low too in concentration. I scrape ceels, lyse cells with lysis buffer having NP-40, centrifuge at 5000rpm.. Take the supernatant and load on the gel.
Can someone suggest a better protocol?
Please do.
Thankyou

Do you know anything about your antibody? Has it been used successfully for Westerns in other labs? Was it raised against a synthetic peptide? Does it work on whole cells?

GPCRs are notoriously low in concentration and also have a lot of secondary structure, so it is quite possible that the epitope for your antibody is not intact, especially if it was raised against a synthetic peptide. You might try running a non-denaturing gel, or try several different antibodies, preferably a polyclonal one raised against the whole protein (although these have their own problems.)

Did you boiled the sample before loading the gel?
Usually GPCRs aggregate when the sample is boiled. Try both ways.
Also consider to perform WB on enriched membrane fraction (e.g. spin at 40000 x g) and as a "negative" control on cytosol. Good luck

val said:

Did you boiled the sample before loading the gel? Usually GPCRs aggregate when the sample is boiled. Try both ways. Also consider to perform WB on enriched membrane fraction (e.g. spin at 40000 x g) and as a "negative" control on cytosol. Good luck

I heated the sample to 80 degrees for 5mins before loading. I didnot boil. I will try both this time. Thanks