protein pptn in acetone problem

John A. Newitt newitt at nih.gov
Tue Nov 21 14:16:55 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

In article <stefanba-2011951413010001 at mun1.mun.uu.se>,
stefanba at bio.embnet.se (Stefan Bäckström) wrote:
> I have had some problems with precipitating and concentrating a protein
> sample using TCA because it precepitated the detergent (NP-40) in my lysis
> buffer but now I use a methanol/chloroform method which works very well
> for me and gives a pure protein pellet which is easy to dissolve.
> 
> You can find the protocoll in 
> 
> Wessel et al, Analytical Biochemistry 138, 141-143 (1984)

I have also used the excellent and under appreciated MeOH/CHCl3
precipitation procedure of Wessel and Flügge for
precipitating/concentrating proteins from high salt solutions prior to
SDS-PAGE.  I would caution you, however, to take care with phosphate
buffers in the presence of other salts as the phosphate will precipitate
with this procedure under some conditions.  In my case, I was using
solutions containing 50 mM NaPi and 1.0 M NaCl.  Presumably the NaCl
decreased the solubility of the phosphate in the MeOH/CHCl3 mixture.  If
this occurs in your case it is easy to see because the salt crystals
pellet through the CHCl3 layer in the penultimate spin.  Protein will not
do this--it will stay on top of the CHCl3 layer.  Good luck.

Regards,

John A. Newitt, Ph.D.           |   <newitt at nih.gov>
National Institutes of Health   |   FAX: 301-402-0387
Bethesda, Maryland  USA         |   



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