yeast microsome/membrane wash ??

EK anybody at uchicago.edu
Thu Apr 17 23:57:42 EST 2003


"jon" <jonathon at rogers.com> wrote in message
news:3E9D716A.2DDE6354 at rogers.com...
> 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.
>
The sonication could as well be a culprit here, and you can check if this is
the case by lysing the yeast vs.sonicating, as well as by resuspending the
membrane pellets using a pipet or a Vortex. If your protein just has a
membrane anchor, that should not necessarily mean that it is insoluble.
-Emir





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