[Cell-biology] Re: Western blot detection of membrane proteins
Dr Engelbert Buxbaum
(by engelbert_buxbaum from hotmail.com)
Sun Mar 11 10:02:14 EST 2007
Samuel C McNeely wrote:
> I need to detect MRP1, a membrane protein, by western blot and have been
> unable to do so. Do membrane proteins require higher concentrations of
> detergent in order to be soluble in lysis solution? What is the ideal
> lysis buffer? Thanks.
You need an antibody that works in Westerns (that is, against the
denatured protein). Large trans-membrane proteins like ABC-transporters
blot better in thank than in semi-dry blotters, and Dunns buffer (with
50 uM SDS) works better than Towbins. Like with all glycoproteins, you
may consider doing an Eastern rather than a Western blot.
It may also be advantageous not to use a whole cell lysate but to enrich
the plasma membrane by isopycnic centrifugation (table top
ultracentrifuges with 0.2 ml sample tubes are ideal) and then process
that. This limits the number of proteins in the lysate, but requires a
larger sample size for the additional handling. Hypotonically lyse the
cells to ghosts, collect by centrifugation, then potter and crap-spin. A
1-step sucrose gradient (35%) is then used to collect the PM at the
interface between sample and sucrose.
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