Hi, would any of you advise a good buffer to disolve the membrane protein fractions after TCA or methanol precipitation? The buffer should be compatible for Trypsin digestion and will be better to not have any detergent. We have a buffer, 8M Urea/50mM ammonium bicarbonate, for the intracellular proteins, but not sure if it works fine for the membrane proteins.
There's a pretty good chance that the pellet will dissolve in 8M urea. If that doesn't succeed, try guanidine hydrochloride.
use buffer containing compatible detergent such as Deoxycholate or sarcosine ( low concentration 0.5 % w/v) . chances that these will help to solubulize the protein even in absence of urea
Adam, Amulya, thanks for your advices. I told to a colleague who did her PhD on the membrane proteins and she said she could only dissolve it in HEPES solution with the existence of SDS. I think I will try the urea buffer first as it is compatible for the followed MS analysis, otherwise, I will see if the reagents you suggested can help in my case.
You could dissolve the pellet in SDS-PAGE sample buffer, run it on a gel, do an in-gel trypsin digestion, and run the digest on LC-MS.
See this paper for the technique:
http://www.ncbi.nlm.nih.gov/pubmed/17406544
Thanks, Adam. Yes, we had a protocol of this process. But we tried to avoid the SDS-PAGE step as it is time consuming. We also try to avoid adding detergent into the dissolved buffer as it will require us to order another expensive Ziptip cleanup tips. The method I am using is SWATH-MS, and if I can successfully dissolve it into a non-detergent-contained buffer, it will save me a lot of efforts. I will try to do some preliminary tests and see what i can get. will update my results later here.
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