Make sure to use the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation if the substrate is a crude lysate.
If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 µM.
1-2 µl of enzyme is a good starting point for a 1 hr incubation of 1µg of protein.
The consensus sequence is substrate-dependent. In some target proteins, prior phosphorylation is required, but a general recognition sequence has not yet been elucidated