Mitogen Activated Protein Kinase Kinase 2 (MAP2K2)
This is a two-step assay where inactive MAPK (0.06 mg/ml) is activated by MKK2 (diluted in 25 mM Tris, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.01% Brij35, 1 mg/ml BSA) in 25.5 µl containing 25 mM Tris, 0.1 mM EGTA, 0.01% Brij35, 10 mM magnesium acetate and 0.005 mM ATP. After incubating at room temperature for 30 min, 5 µl from the first reaction is pipetted into 20 µl of the second reaction mix containing (final concentration) 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.66 mg/ml myelin basic protein (MBP), 10 mM magnesium acetate and 0.05 mM [33P-γ-ATP] (500 -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.
MKK2_6.pdf (90 KB)