mitogen-activated protein kinase 7 (MAPK7)
Co-expression with GST-MKK5 S311D T315D
ERK5 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1% Mercaptoethanol) is assayed against MBP in a final volume of 25.5 µl containing 50mM Tris pH 7.5, 0.1mM EDTA, 10mM DTT, 0.33mg/ml substrate, 10 mM magnesium acetate and 0.05mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated for 30 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.
ERK5_screening.pdf (88.85 KB)