I am working on a GPCR which we have predicted bioinformatically to be expressed in the endoplasmic reticulum. We have transfected HEK 293 cells and we are harvesting cells (24 hours, 48 hours, 72 hours....) for Western blot analyses. I wish to ask if anyone working or has worked on such before can let me know the protocol they used, or if any one can suggest a suitable protocol? Thanks for the suggestions.
Sometimes it is very difficult to resolubilise membrane proteins. You can try with a saturated solution (6 M) of guanidine thiocyanate and once your protein is in solution, you can precipitate it with acetone, spin down (not too long), wash several times with 70% ethanol, spin again and then try to resuspend the pellet in your SDS buffer.
I would start with the standard western blot protocol with RIPA buffer.
Make sure not to boil the sample since membrane protein tend to aggregate after boiling.
Agree with Jian: be careful with boiling the samples since with MP it may induce aggregation. We use SDS-buffer combined with urea and incubate 10 min at 50 oC before SDS-PAGE and WB. Good luck, M
There are plenty of ways to solubilize membrane proteins using detergents of strong to mild capacity. SDS-PAGE buffer without boiling the sample and RIPA buffer during western are the preferred ways to isolate membrane bound proteins.
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