2D-PAGE of G protein coupled receptor - Need advice on sample preparation - GPCR overexpressed in HEK293 cells (Jan/10/2011 )
I know this is a long shot, the literature on this subject is limited for a reason, 2D-PAGE on multipass membrane proteins is really difficult but my supervisor wants me to give it a try. My protein of interest is a GPCR which I over express in HEK293 cells. I get quite good resolution of this receptor on SDS-PAGE PAGE/western blotting when I lyse my cells in PBS + 1% B-dodecyl-N-maltoside (a non-ionic detergent). I dilute my lysates in conventional 2x Laemmli sample buffer and heat them to 65 degrees prior to loading (boiling the samples causes the protein to aggregate at the top of the gel). The receptor has extensive N-linked glycosylation.
The reason for trying 2D-PAGE is that I see interesting shifts in molecular weight for my receptor in the presence of an interacting protein and my supervisor thinks that 2D PAGE might help us to narrow down the mechanism behind these shifts. She wants me to express the receptor with and without the interacting protein, do a 2D separation and then western blot for the receptor, I already know that Coomassie staining will not be sensitive enough. I understand that isoelectric focusing of membrane proteins is challenging because they aggregate/precipitate under aqueous conditions. I think this will be even more of a problem for a protein with 7TM domains! Is there a proteomics guru out there who has successfully separated proteins with multiple TM domains and can advise me on a starting point for appropriate buffer conditions? Or tell me that I'm wasting my time even attempting this?
if you do 2d then don't dilute the lysate with laemmli sample buffer.
you can add non-ionic or zwitterionic detergents or urea (or both) to the ief gel to maintain solubility.
download this handbook from ge healthcare.