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
