CIP and protein - (Jan/24/2007 )

Hi everyone,
I'd like to incubate my protein lysate with some CIP (phospatase), run a western blot and see if my band of interest (I think that band is a post-translational modification) disappear. Which buffer I should use to extract and preserve protein that work as well as a CIP buffer? I should IP my protein and then incubate with the CIP and beads?...anyone ever try to set up an experiment like this one and have some suggestion to give to me?
thnks for your collaboration

Mario

since it is an alkaline phosphatase, you should use a buffer with a pH above 7 (8.0 should be okay).

you should see the band shift, not disappear outright, unless you are probing for phosphorylated proteins.

Thanks for the suggestion...I will try it.
what i m doing is.. diluited my extraction buffer (it's urea buffer....ouch!!!) with a CIP buffer+ proteinase inhibitor+excess of enzime, check the ph incubate 1 h at 37....I'm running the gel right now....I will tell you how will come

thanks
Mario degree