IUBio

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)
>wrote:
>
>> 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




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