yeast microsome/membrane wash ??

Kyle Legate legatek at sympatico.ca
Wed Apr 16 19:09:38 EST 2003


jon wrote:
> 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.
>
Are you sure it's an integral membrane protein? It could be a tightly bound
peripheral protein that partially releases in the salt conditions you are
using. See the mammalian SRP receptor for an example of a protein that was
assumed to be integral for many years. It's peripheral after all (since
1986).

Assuming it is integral, is it a native protein, or are you overexpressing
it? Overexpression of membrane proteins (especially ones that demonstrate
saturable binding--search pubmed for papers by Kim PK and Andrews DW) could
result in the yeast machinery becoming overloaded, with the overflow
remaining in the cytosol.





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