yeast microsome/membrane wash ??

jon jonathon at rogers.com
Wed Apr 16 10:16:43 EST 2003


I am trying to purify a integral membrane protein in yeast.  I've
isolated yeast
microsomes/membrane fraction and proceeded to wash the membrane pellet
with a high
salt Tris buffer(no detergent).  I spun down at 150,000Xg and saved the
supernatant
(for analysis).  I then solubilized the washed membrane pellet with
triton detergent, I
then spun down  any insoluble material.  I collected the solubilized
supernatant.

I did a Western running the wash supernatant and solubilized supernatant
and it seems
that my integral membrane protein I'm trying to purify appears in the
wash supernatant
at an equal concentration to my solubilized supernatant.

Does one expect to find so much of the protein (especially integral
membrane proteins)
being lost in the washing of membranes?  Shouldn't it all have been
pelleted down.

Note: I resuspended the membrane pellets by sonication, would this have
any influence
on my observed results on western.


Thanks

Jon



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