In vitro kinase activity assay

Kinases were immunoprecipitated from transfected HEK293T cells. After washed in wash buffer (200 mM NaCl, 40 mM HEPES pH 7.5) and kinase assay buffer (50 mM potassium acetate, 30 mM HEPES, 5 mM MgCl₂), purified kinases were incubated in 45 μl kinase assay buffer in the presence of 200 μM ATP-γ-S and 20 μM ATP at 30°C for 1 hr with vortex. 2 μl EDTA (0.5 M) was used to stop reaction followed by additional 2 μl PNBM (50 mM) to start alkylation for 30 min at room temperature. Assay was completed by boiling reaction mixture at 95°C for 5 min with SDS loading buffer.