descrepancy between total cell lysate and cell fractionation - (Dec/19/2010 )

Hi All,

I have done total protein lysates using RIPA buffer(containing Igapal, SDS, deoxycholate etc) and observed an inhibition of growth factor induced phosphorylation by my treatment. Generally, my protein should translocate to nucleus upon growth factor addition. Thus I expected inhibition of this process with treament of my inhibitor compound. However, translocation was exactly the same. All of the phosphorylated protein was in the nuclear fraction as expected but no difference, the kinetics were the same over a number of timepoints.

I have been thinking of doing IF to confirm inhibition rather than subcellular fractionation. But I was wondering if this is an issue with my nuclear extracts? I used tubulin and lamin A/C and confirmed fractionation. Could I not be isolating something from the nuclear extract like histone bound proteins? THe method I employed involved using sucrose and triton x for cytosolic and high salt(o.42M NaCl) for nuclear. I dont sonicate or dounce my cells.

Ideas?

what do you mean by IF? Do you think in terms of immunocytochemistry? this would be a useful alternative if you have appropriate antibodies...