Commercial recombinant Akt preparations show different specificity. A, S10 phosphorylation by recombinant Akt1(prep2; see Methods). Recombinant Akt1(prep2) was incubated with His-tagged p27 in the presence of non-labeled ATP. Akt inhibitor was added where indicated. Samples were separated by SDS/PAGE and analyzed by western blot with the indicated antibodies. B, Radiolabeled phosphate incorporation by Akt1(prep2). Reaction was carried out as described in Methods. Autoradiograph shows phosphorylation of GSK, hwtp27 and p27S10A. C, Akt1(prep2) lacks specificity. Kinase reaction as described in B was performed in the presence or absence or bovine serum albumin (BSA). Autoradiograph shows Akt1(prep2) phosphorylates BSA as well as p27. D, Radiolabeled phosphate incorporation by Akt1(prep3). Kinase reaction performed as in B with recombinant Akt1(prep3; see Methods). E, Phospho-amino acid analysis of hwtp27 and p27S10A phosphorylated by Akt1(prep3). Phosphorylated p27 was subjected to PAA as described in Methods. Upper panel: schematic representation of phospho-amino acid migration. Middle panel: PAA corresponding to hwtp27. Bottom panel: PAA corresponding to p27S10A.