Membrane protein isolation for western blotting

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Mon Jan 25 11:38:19 EST 1999

In article <pxpst2-2501991015150001 at pelli.pathology.pitt.edu>, pxpst2 at unixs.cis.pitt.edu (Peter) wrote:
>In article <78gq9q$9je$1 at panix3.panix.com>, iayork at panix.com (Ian A. York)
>> Disrupt the cells mechanically (traditionally douncing works) and
>> centrifuge first at low speed (to get rid of nuclei, if you want to do so)
>> and then at around 100,00g (if I recall correctly) for an hour or so.  The
>> membranes pellet.
>This is DEFINITELY the easiest way to to enrich for membranes.  

Not very high yeild however (lots of memebrane stay with nuclei
and sheared cells) and does not enrich for ALL membranes equally.
Here is what I do (somewhat paranoid): freeze in LN2 ->  thaw --> 
dounce --> pellet 40Kxg, 30' --> resuspend, dounce --> pellet --> 
dissolve in 1% Triton X-100 --> pellet --> sup contains representative 
collection of all membrane proteins (nuclei do not dissolve). 

        - Dima

More information about the Proteins mailing list

Send comments to us at biosci-help [At] net.bio.net