Yeast-Protein-extraction and following identification of a nuclear protein

J. Martinez-Irujo jjmirujo at unav.es
Thu Nov 11 05:26:57 EST 1999



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 in the
> following buffer:
> (glass bead disruption buffer)
> 20 mM     Tris-Cl, pH 7.9
> 10 mM     MgCl2
> 1 mM        EDTA
> 5%(v/v)    Glycerol
> 1 mM        DTT
> 0.3 M       ammonium sulfate
> no protease inhibitors
>
> After disruption (checked by microscope) I spinned down the debris,
> and used the supernatant for SDS-PAGE and blotting.
> No signal was recieved from this extraction.
>
> But when i treated the debris from above with urea buffer (8 M urea, 100
> mM Na2HPO4, 10 mM Tris-Cl, pH 8), centrifuged, an used this supernatant
> for western-blotting i got a weak signal.
>
> Questions:
> Why is my target protein not in the first supernatant?
> Is the nucleus from yeast cells destroyed by  the glass-bead + vortex
> method? (my target protein is in the nucleus)
> My target protein is a DNA-binding protein, is it stripped off  from DNA
> by the glass-bead disruption buffer mentioned above?
> If not, what buffer would be suitable?
> Do you know a better method for small-scale protein extraction from
> yeast (especially for a DNA-binding protein)?
>
> Thanks, Lutz

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).
Good luck.

--
Juan J. Martínez Irujo

Departamento de Bioquimica
Universidad de Navarra
Pamplona, Spain







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