Fig. 1. A time course kinase reaction.

(A) A master kinase reaction mixture is prepared by adding kinase buffer, purified protein, and ATP. Before starting the reaction, add kinase and [γ-³²P] ATP last. Incubate the master mixture in a heat block set at a desired temperature depending on the kinase. At each time point, remove 10 µl of the master reaction mixture, transfer to a new tube containing 3x Laemmli sample buffer to stop the reaction, and keep the tubes on ice until all the samples are collected.

(B) Left: when running a SDS-PAGE to separate phosphorylated proteins from unincorporated [γ-³²P] ATP, stop running the gel before the dye front runs through the gel. Trim the gel to remove the stacking gel and the dye front. Middle and right: stain the trimmed gel with Coomassie blue solution and destain until desired contrast is reached.