In-gel kinase assay

Frozen seedlings were ground in liquid nitrogen with mortar and pestle and sonicated three times for 20 s in the extraction buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA 50 mM β-glycerophosphate, 100 μM Na₃VO₄, 2 mM dithiothreitol [DTT], Complete protease inhibitor cocktail Roche) using approximately 0.5 mL of the extraction buffer for each 1 μg of plant material. After sonication, the extracts were centrifuged at 18,000 rcf for 30 min at 4°C, and the supernatants were used for further studies. In-gel kinase activity assays were performed using a method described previously (Wawer et al., 2010). Briefly, protein samples were separated in 12% SDS/PAGE gels with 0.5 mg/ml histone embedded in the separating gel as a kinase substrate. After electrophoresis, SDS was removed by washing in washing buffer (25 mM Tris/HCl, pH 7.5, 5 mM sodium fluoride, 0.5 mg/ml BSA, 0.1% Triton X-100, 0.5 mM DTT and 0.1 mM sodium orthovanadate) three times each for 30 min at room temperature. Proteins were renaturated overnight in renaturing buffer (25 mM Tris/HCl, pH 7.5, 5 mM sodium fluoride, 0.1% Triton X-100, 1 mM DTT and 0.1 mM sodium orthovanadate) at 4°C with three changes of buffer. The gel was incubated for 1.5 h at room temperature in 10 ml of reaction buffer (10 mM Tris/HCl, pH 7.5, 2 mM DTT, 0.1 mM EGTA, 15 mM MgCl2 and 20 μM ATP, supplemented with 50 μCi of [γ-³²P]ATP). Unincorporated [γ-32P]ATP was removed by extensive washing in 5% trichloroacetic acid with 1% sodium phosphate. The gels were stained with Coomassie Brilliant Blue R250, dried and exposed to autoradiography.