acetone precipitation of samples (for SDS PAGEs)

Doug Easton dpeaston at wzrd.com
Mon Nov 18 22:36:28 EST 1996


siyer at bmg.bhs.uab.edu (Sai) wrote:

>hey all, 
>            i've posted this before, but i figured i'd try again to see if
>there were any other ideas.  i've been doing acetone precipitation to
>remove salts from my samples prior to loading on sds gels.  the basic
>protocol is add 8 vol. of acetone to 1 vol. of protein sample --->
>precipitate at -20 degrees for 4-6 hours ---> spin down for 20 min at
>14,000g (max speed on centrifuge) at 4 degrees to pellet out the
>precipitated protein ----> decant the supernatant and immediately
>solubilize the pellet in sds loading buffer.  the problem is see is that i
>still get streaks in my gels.  theoretically, acetone is an uncharged
>organic solvent and so should not create any distortions in the gel cuz
>its not charged.  so if there were any residual acetone left, that still
>shouldnt create the streaks...yet i still see streaks.  anybody have any
>ideas on this?  or other methods to remove salts from samples (i.e tca
>precipitations etc) would also be welcome...thx...cheers....

>-- 
>Sai Iyer
>siyer at bmg.bhs.uab.edu

I expect that your streaks are caused by some protein precipoitate
which has not disolved in the SDS Buffer. If you are confident that
the resoluabilisation is not selective, you could clean up the samples
with a brief high speed spin (10 K 10 min.).

-Doug Easton
>graduate student; dept of biochemistry and molecular genetics
>university of alabama at birmingham





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