protein pptn in acetone problem

Stefan Bäckström stefanba at bio.embnet.se
Mon Nov 20 08:13:01 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,
> 
> 
> 
> Brett Lindenbach
>     
> Program in Immunology                              
> Washington University - St Louis                  
> brett at borcim.wustl.edu                             
> 
> "I own my own pet virus. I get to pet and name her." - Cobain

Hello Brett.

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)

"A method for the Quantitative Recovery of Protein in Dilute Solution in
the Presence of Detergents and Lipids"

4 vol of methanol is added to sample. 
vortex briefly
1 vol of chloroform is added.(is dissolved in the methanol. No phase separation)
vortex briefly
3 vol of water (Gives phase separation. Methanol+water in one and chloro in one)
vortex vigorously
Centrifugate 1 min 9000 g (I use full speed in microcentrifuge)
Upper phase is discarded (protein is in the interphase)
3 vol of methanol is added (methanol + chloroform is mixed)
Centrifugate 2 min at 9000 g (pellets the protein)
Discard the methanol/chloroform
Dry in speedvac (both the methanol and the chloroform evaporates)

Ready to dissolve..

If I have 1 ml of sample to ppt, I use five 2 ml tubes and during the last
centrifugation I collect all the protein in one tube before the drying.



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