? on protein extraction

John A. Newitt newitt at removeme.nih.gov
Fri Jun 25 12:03:13 EST 1999


In article <376FA782.6A0E75E0 at pitt.edu>, Ruben Zamora <zamorar at pitt.edu> wrote:

> Hello,
> 
> I would like to isolate protein from cultured cells to perform Western
> blot. What is the best method for extraction, to lyse the cells by
> adding Triton X-100 to the buffer or just leaving Triton X-100 out the
> extraction buffer and completing 4 or 5 freeze-thaw cycles?

I would avoid freeze-thaw since proteases may chew up your protein before
you can extract it.

I used to use an extraction buffer containing 6M guanidine HCl, 250 mM
TrisHCl pH 8.8, 10 mM EDTA, and 2% (v/v) mercaptoethanol (include 1mM PMSF
fresh if you want, but probably isn't necessary). You can added it directly
to the culture dish (after a PBS wash), scrape, and pipette into a tube.
Sonicate (nucleic acids are also extracted, so the samples are very viscous
initially). Work up an aliquot for SDS-PAGE by MeOH/CHCl3 precipitation
(Wessel and Flugge 1984 Anal. Biochem. 138:141-143) to remove the guanidine
and salts.

This protocol has the advantages that the rapid denaturation by the
guanidine should prevent proteolysis (prevented degradation of proteins in
solubilzed sperm whereas an SDS-solubilization buffer was ineffective) and
that samples in the guanidine sol'n can be stored for quite a long time at
-20 degrees C with no obvious changes in the polypeptide profile. If you
don't want total protein or need to load a lot of your particular protein
in each lane it may not be right for you since I found that approx. 1-5 X
10^5 cells per lane was the max I could load on full size gels (Hoefer
SE600, 15 lanes) due to protein and nucleic acid overloading
considerations. Hope this helps.

Regards,

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

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