In vivo, my ATP competitive inhibitor works nicely and known substrates were confirmed by both western blotting and phosphoproteomics. However when I perform an in vitro kinase assay, I see nice phosphorylation with 32P-ATP but when I add the inhibitor, the latter does not seem to inhibit the phosphorylation, also not on the known substrates. I've tested a whole range of concentrations and it works only at very high non-physiological concentrations. Recombinant substrate was mixed with the purified kinase complex that was added bound to streptavidin-sepharose beads.

Any suggestions?