acetone precipitation of samples (for SDS PAGEs)

Wulf Dirk Leuschner wdl at gen.mpib-tuebingen.mpg.de
Thu Nov 21 13:53:24 EST 1996


cohlberg at csulb.edu (Jeff Cohlberg) wrote:

>: The reference is Anal. Bioch. 138:141-143. I have found this to be *much*
>: better than TCA or acetone precipitation. For very dilute solutions I have
>: added carrier proteins to ensure absolute recovery.

>I heartily agree.  We recently started using this method.  We always get
>virtually 100% recovery and never have any trouble redissolving the
>precipitate.

> -- 
>Jeffrey A. Cohlberg, Professor
>Department of Chemistry and Biochemistry
>California State University, Long Beach
>1250 Bellflower Boulevard, Long Beach, CA 90840
>Phone: (310) 985-4944	FAX: (310) 985-8557   E-mail: cohlberg at csulb.edu

Hi Jeff,

this really surprises me. Do you use this precipitation technique with
solubilized membrane proteins? I have got a lot of problems with
resolubilizing the pellet in various sample buffers (for SDS-PAGE).
Adding 6 M urea to the sample buffer does not change much.
 In fact, sometimes the resolubilization is o.k. But I would like to
have reproducible results concerning the efficiency of protein
recovery from the precipitate.
Using different detergents does not lead to different results...
Air-drying the pellet is no better than drying it in the speed-vac...
If you have no problems in redissolving your pellet, what do you think
does the trick? I'd be very glad if you came up with an answer!

By the way, did you ever try PEG precipitation? If so, what kind of
PEG do you use (PEG400 or 4000 or ...). In what concentration range?
Does it work well with detergent solutions?

WDL.




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