Yeast-Protein-extraction and following identification of a nuclear
C.Gaenzler at dkfz-heidelberg.de
Mon Nov 15 05:20:12 EST 1999
" J. Martinez-Irujo" wrote:
> Lutz Thon wrote:
> > I am trying to detect a nuclear protein from Sacc. cerevisiae by a
> > western-blot.
> > I used a glass-bead + vortex method to disrupt the cells
> > Questions:
> > Why is my target protein not in the first supernatant?
> > Do you know a better method for small-scale protein extraction from
> > yeast (especially for a DNA-binding protein)?
> I would try other disruption methods such as a French press or a sonifier.
> The buffer seems to be appropriate (amonium sulfate will prevent proteins
> from binding to DNA, though I would add a cocktail of protease inhibitors).
I work with S.pombe but things should be similar. I break up the cells
by french press in a 4M Urea buffer and my protein is still not soluble
(but many others are) so in this first step after centrifugation it is
already a good purification step! Then I can go on with 6M guanidinium
HCl or higher molarity Urea and get the protein into solution. Further
purification steps are the appropriate columns like hydroxy apatite and
ion exchangers as described in: Braspenning et al., 1997, A general
purification protocol for E7 proteins from "high- and low-risk" human
papillomavirus types expressed in the yeast Schizosaccharomyces pombe.
German Cancer Research Center (DKFZ)
Applied Tumorvirology - ATV F0200
EMail: c.gaenzler at dkfz-heidelberg.de
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