Protein yeast extraction for Western blot - (Mar/23/2006 )

Hi,

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??

Thank you!

-paulinahe-

I've once extracted protein from P. pastoris with glass beads.

See page 14 of the following link

http://www.invitrogen.com/content/sfs/manuals/ppicz_man.pdf

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.

-aspergillie-

Thanks,

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...

-paulinahe-

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).

-mdfenko-