Lutz Thon schrieb:
> 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
Hello Lutz,
I could detect an yeast transcription factor (which binds constitutively to
DNA) using glass beads and the following lysis buffer.
1 X PBS containing 1mM EDTA, 5% Glycerol, 0.1% NP-40 and
protease inhibitor.
The isolated protein could be succesfully used for band shift assay. I'm not
sure whether the 'NP-40' is an essential component, but it may help you.
Byung-Hoon
Zentrum fuer MolekularBiologie der Pflanzen
Uni Tuebingen
