ammonium sulphate ppt of membrane protein

Emir Khatipov khatipovNO at NOuchicago.edu
Tue Apr 23 17:02:01 EST 2002


If you mean your protein floats as a non-soluble layer on top of the buffer
with ammonium sulfate, then it is normal for the situation like yours.
Pelleting occurs when the density of the precipitated material is higher
than that of the buffer. Your case is similar to centrifuging milk: lipids
float, denser proteins (if ever)precipitate. You solubilized the lipids with
triton and then increased density of the solution by adding the salt. That
resulted in separation of the lipids. You should probably use (cold?)
acetone precipitation, or treat the solubilized membranes with lipase.
However, you protein might become unstable without lipids. I would try
acetone first.
Emir


"rajneeli" <rajneeli at lebs.cnrs-gif.fr> wrote in message
news:a315f5c3.0204230827.93e74ba at posting.google.com...
> Hi Netters
>
> I have used ammonium sulphate for the precipation of membrane bound
> protein after extraction with triton X-100. after centrifugation, I
> have yellow layer (my protein is yellow in color) on top without
> pelleting at bottom. Could any body know why there is no pelleting
> after centrifugation at 18000 rpm even after ammonium sulphate ppt.
>
> thanks in advance
>
> raj





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