Yeast-Protein-extraction and following identification of a nuclear protein
stu32394 at mail.uni-kiel.d400.de
Wed Nov 10 11:23:36 EST 1999
I am trying to detect a nuclear protein from Sacc. cerevisiae by a
I used a glass-bead + vortex method to disrupt the cells in the
(glass bead disruption buffer)
20 mM Tris-Cl, pH 7.9
10 mM MgCl2
1 mM EDTA
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.
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)?
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