membrane preps from cultured mammalian cells

JLCruise jlcruise at stthomas.edu
Tue Oct 8 17:23:04 EST 1996


I am looking for suggested methods of making rapid membrane preps from
various mammalian cells in culture.

The uses I have in mind for these preps are 1) receptor-binding studies
and 2) western blotting of membrane-associated proteins.  Most of the
lines I am using are adherent.  While I have experience with standard
protocols (modified Neville method, Prpic membrane prep, crude microsomal
preps) that work well for me when I start with tissues, my limited
experience with cell cultures is of low yield and probably cleaner preps
than I may need.  Searching the literature on adrenergic receptor studies
(which is what I'm doing the binding assays on) has produced the full
spectrum from hypotonic lysates without ANY fractionation to very pure
plasma membrane fractions produced by long protocols. 

Can anyone recommend a rapid, fairly simple membrane prep with decent
yield from cultures that they know to produce membranes suitable for
adrenergic (or other) binding studies? 
For the westerns, my need is merely for a prep with good yield, that is
clearly membrane-enriched, relative to total cell lysate.

Any tips will be appreciated.  Please email me directly or post here. 
Thanks.


jlcruise at stthomas.edu

Jennifer L. Cruise
Dept. Biology
Univ. St. Thomas
St. Paul, MN



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