protein pptn in acetone problem

Stephen R. Lasky Stephen_Lasky at brown.edu
Wed Nov 22 07:24:01 EST 1995


In article <199511171834.MAA18089 at borcim.wustl.edu>,
brett at BORCIM.WUSTL.EDU (brett) wrote:

> Hello, I'm trying to ppt. my protein using cold acetone. It was in a 50uM
> sodium phosphate buffer. I added 8 vol. acetone, and now have a large,
> chunky ppt. I believe the large chunks are phosphate, as buffer alone gave
> the
> same appearance. Parallel ppt's of the same protein in a citrate buffer
> gave a finer, milky ppt. I now want to dry down the pellets and resuspend
> them for SDS-PAGE analysis, but am worried about the salt. Anyone have any
> suggestions for what to do? I thought I might resuspend the chunky pellets,
> dialyze into another buffer, and reppt. Thanks in advance,
> 
> 

Try using 3 volumes of ice cold acetone, overnight at -20 deg C instead of
8 volumes.  This worked for me but I wasn't in a Phosphate buffer.  If you
get bad gels and a lot of streaking or ppt of the salt in your loading
buffer, you can dialyze out the salt in a microdialysis aparatus.

SRLasky

-- 
Stephen R. Lasky Ph.D.
Section of Experimental Therapeutics
Roger Williams Medical Center
Providence, RI 02908
Phone: 401-456-5672      Fax: 401-456-6569
e:mail: Stephen_Lasky at brown.edu



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