To further investigate immobilized PKA activity, we incubated beads with HeLa cell lysates (Fig. ). After only one hour of incubation time at room temperature, we observed significant phosphorylation of endogenous PKA substrate proteins across a wide range of molecular weights. Importantly, the pattern of phosphorylation observed on the western blot for the immobilized PKA (Fig. Lane 1) compared favorably with that of the free PKAcs enzyme used as a positive control (Fig. Lane 5). As before, the experiment containing immobilized PKA (Lane 1; 50 µL of bead suspension), approximately 67 µg of Halo-PKA fusion are present in bead-bound form; this provides >70% of the VASP phosphorylation afforded by 500 ng of nonimmobilized PKAcs, whereas no PKA substrate phosphorylation over background was detected with 500 ng of non-immobilized Halo-PKA (Lane 4).