MAPK-activated protein kinase-1a
PDK1 and ERK2
MAPKAP-K1a/RSK1 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% β-mercaptoethanol, 1mg/ml BSA) is assayed against KKLNRTLSVA in a final volume of 25.5 µl containing 50 mM Na-b-glycerophosphate pH 7.5, 0.5 mM EDTA, 30 µM substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated for 40 min at room temperature. Assays are stopped by addition of 5 µl of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.
RSK1_4.pdf (87.93 KB)