I have been trying for a long time to see by Western Blot a 450 kDa-peripheral membrane protein from the Golgi apparatus with no success. I know that the electrophoresis and the membrane transfer is not problem, since I can see the protein ladder that goes up to 460 kDa. I think I am loosing the protein during the lysis...My protein is highly hydrophobic and insoluble and goes to the pellet. This is the protocol I have been using (with small modifications each time):
- Lyse the cells with NP-40 or RIPA buffer (usually a 15cm dish in 500ul of buffer)
- centrifuge at 10.000g to remove the soluble fraction
- resuspend the pellet in the same lysis buffer+6M urea
- Sonicate in a water bath
- centrifuge 30' at 100.000g
- Collect the supernatant, and run in a 6% gel.
After this I get very little protein in the pellet fraction, one can sometimes see a faint band but considering how much protein I am using it should be bigger. I must be loosing the protein at some point.
I would appreciate very much any tip or comment that could help me to solve this problem.
Thanks a lot in advance!
Its a real challenge to get large TM proteins out and analyzed! An observation from some work I've done with Na/K ATPase is that if the SDS-PAGE samples are heated too high the protein completely disappears from the gel; one need only to gently warm the loading buffer/reducing agent before loading. Almost everyone new tried a traditional boiling sample prep and came away with no bands, Good luck!
Since you are interested in a peripheral membrane protein, I suggest you check in the soluble fraction, because the RIPA buffer, which contains detergent, may have released the protein from the membrane and kept it in solution.
I agree with both Edward Michelini and Adam B Shapiro in addition I'd suggest doing the lysis directly in SDS-sample buffer. After gentle heating, briefly spin down undissolved material (there shouldn't be much) and use the supernatant.
If you still have problems, sometimes CTAB-PAGE followed by eastern-blotting (doi:10.1016/S0003-2697(02)00639-5) works better than SDS-PAGE, especially with membrane proteins.
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