ab62291 was diluted to 20ng/μl with assay dilution buffer (4 mM MOPS,pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT), followed by 2-fold serial dilutions, and then the 10μl diluted proteins were used to phosphorylate the S6K substrate peptide(CKRRRLASLR) in the following assay condition: 10 μl diluted ab62291 10 μl S6K substrate peptide (1 mg/ml stock) 5 μl [32P] ATP mixture (250 μM ATP, 0.16 μCi/μl in 4x assay dilution buffer) The various reaction components, except [32P] ATP, were incubated at 30 C and the reaction started by the addition of [32P] ATP. After 15 minutes, the reaction was terminated by spotting 20 μl of the reaction mixture onto a phosphocellulose P81 paper. The P81 paper was dried and washed several times in 1% phosphoric acid prior to counting in the presence of scintillation fluid in a scintillation counter. The actual counts, using various dilutions of the enzyme in the assay, are shown in graph above. ab62291 Kinase activity plo