I am trying to characterize a so far unknown membrane protein (a GPCR). I overexpressed it in HEK cells and want to verify its abundance via WB. So far there is one problem: WB analysis shows more than one band (could be aggregates, since the bands are 1x, 2x and approx 4x the protein size) and the signal is not increased in cells overexpressing the protein compared to controls. I checked for transscription, and i was able to show a higher mRNA concentration for transfected cells. So either my protein does not make it to the PM or the Ab does not work or my membrane/lysis protocol is not optimal. Does anyone have experiences with GPCR membrane extration and might be willing to share the protocol? Thank you!
Antibodies for GPCRs are almost universally poor. If you want a reliable blot for a GPCR, you can use an N- or C- terminal tag, such as FLAG, 1D4, etc... that has a well characterised/validated antibody.
The larger bands you see are not likely to be aggregates. These should be dissociated by the loading buffer you are using to run the gel. Alternatively they could be receptors with post-translational modifications, although 4X sounds a little extreme. Are you running a non-transfected or mock-transfected control on your gels as well?
Thank you so much for your answers so far. I had the same thoughts and I am already cloning a tag to the construct.
Above mentioned results were obtained using RIPA lysis buffer used directly on the cell culture plate followed by burst-sonification on ice.
I was advised to try Urea-containing lysis buffer 6M Urea / 20mM Na-Phosphat/ 0.5M NaCl / 20mM Imidazol / 5mM DTT; but i have several concerns: No detergent is present, so membrane proteins will not solubilize. GPCRs should not be treated with reducing agents since it can destabilize them.
1. Many GPCRs show up as oligomers by SDS-PAGE/western blot.
2. Use a buffer with detergent. DDM is a good starting choice.
3. Are you trying to recover functional GPCRs? If so, you should include a ligand binding assay. That will also tell you whether your receptors are being successfully processed into the membrane.
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