mitogen-activated protein kinase 8
5-20mU of ERK8 (diluted in 50mM Tris (pH 7.5), 0.1mM EGTA, 1mg/ml BSA, 0.1%,β-mercaptoethanol) is assayed against MBP in a final volume of 25.5µl containing 50mM Tris (pH 7.5), 0.1mM EGTA, 0.1%, β-mercaptoethanol, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.005 mM [33P-γ-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5µl of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.
ERK8_3.pdf (81.68 KB)