yeast-protein-extraction and following identification of a nuclear

Lutz Thon stu32394 at mail.uni-kiel.d400.de
Wed Nov 10 11:44:57 EST 1999


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





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