"Greg Pankhurst" <g.pankhurst at spamblock.cfi.unsw.edu.spamblock.au> wrote in
message
news:1fzj4ez.129n0zksj90qoN%g.pankhurst at spamblock.cfi.unsw.edu.spamblock.au...
> Hey All
>> I'm interested in analysing membrane proteins of various cell lines via
> RP-HPLC. The plan was to mechanically lyse the cells, spin down the
> membranes, solublise them and then do the analysis.
>> The problem is getting rid of all the lipid from the membranes. My guess
> is if I inject all the lipid onto the column, all I'll succeed in doing
> is binding a heap of muck to my column. I could do a TCA precipitation
> of my proteins after the solubisation, problem being I'm trying to
> preserve the oxidation/post translational modification state of my
> proteins, and TCA precips seem cause any number of nasty reactions in my
> proteins of interest.
>> Anyone got any bright ideas as to what I might try ??
>> Cheers
> GregP
I am sorry to be a bit sarcastic, but are there any papers on the subject
that would suggest the protocol? In addition, I believe many of membrane
proteins would be lipoproteins or the ones dependent on the membrane for
stability, thus you might be just better off trying to solubilize the
membrane fraction in organic solvents compatible with RP-HPLC, like
acetonitrile, acetone, etc. Also, you should always use a pre-column, so
that the expensive column will not get clogged by gunk. If you still prefer
water-based buffers, try digesting lipids with lipase. However, many
membrane proteins, again, might become unstable and would precipitate.
Anyway, I would go with organic solvents. I am sure you could find a lot on
the subject in Pubmed or in books. There is a number of methods books
specifically dealing with analysis of membrane proteins. And I must say,
this is a very broad and well studied area of biochemistry, so you might
need to look for information pertinent to your particular organism or type
of membranes.
-Emir
