IUBio

Delipidation of Membrane Fraction for HPLC Analysis

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Fri Aug 15 05:52:39 EST 2003


Greg Pankhurst wrote:

> 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. 

Removal of lipids from membrane proteins usually results in aggregation,
the resulting pellet is difficult if not impossible to redissolve. 

The usual procedure is to solubilise the membranes in detergent (Triton,
C12E6, octyl-glucoside, dodecyl-maltoside or similar) and then use
normal chromatographic techniques to separate the
detergent/lipid/protein micelles. As long as you have enough detergent
present that each micell contains at most one protein molecule, this
works very well. Look for a couple of papers written by J.L. Rigaud for
technical details.

Note that RPC is not normally useful even for soluble proteins, as they
can not be eluted from the column again. Only peptides are amendable to
this technique. You may consider hydrophobic interaction chromatography
(HIC) instead, which uses phenyl- or butyl groups instead of octadecyl-
to limit the strength of interaction with the column, but for membrane
proteins even that may not suffice.



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