Protein yeast extraction for Western blot - (Mar/23/2006 )
did someone extract the proteins from yeast and than meausured the concentraction for Western blot of them??
Which method for extraction and measurment did you use??
I've once extracted protein from P. pastoris with glass beads.
See page 14 of the following link
I didn't measure the concentration, but I tried some volumes on SDS-PAGE gel and stained with coomassie and took a suitable volume for western blot.
You could measure the concentration with Bradford, Lowry or BCA method, but I don't have much experience with that.
but the problem is that I can not use Bradford or Lowry assay, because my buffer is not compatible with these assays! I use the MM Yeast Two hybrid system Clontech and the cracking buffer for isolation the proteins...
you might be able to tca ppt the protein from a small aliquot of your sample and dissolve in NaOH then continue with the Lowry determination (i believe that this is the original method).