problems with cell stimulation - (May/20/2004 )

When I stimulate cells with agonist and look at the resulting tyrosine phosphorylation, the signal in the 0 timepoint (unstimulated) is always equal to the signal in the "stimulated" timepoints. I have tried serum starvation for 24h or 48 or even 72 hrs without reducing the signal at 0 time. Any other suggestions?

The stimulation time is also important. I used EGF 100ng/ml to stimulate HCT-116 for 10 min, then i got a thick band in WB, but got no result for 15 min just as unstimulated.

Add tyrosine phosphatase inhibitor and do a time course and dose-response studies.