In Vitro Kinase Assay for GFP-Fusion Proteins

The procedure was performed as described in Mazur et al. (2021) (link). In brief, for immunoprecipitation of GFP-fused proteins, 400 µg of crude protein extract from seedlings treated with H₂O₂ or MV was incubated with 10 µL of GFP-Trap^®_A (Chromotek) for 2.5 h with gentle rocking. After intensive washing, agarose beads with bound immunocomplexes were suspended in 20 mM Tris–HCl, pH 7.5 supplemented with 150 mM NaCl and 4 µg of Myelin Basic Protein (Sigma-Aldrich) per sample. To each sample, ATP supplemented with 1 μCi of [γ³²P]ATP in kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM EGTA, 1 mM DTT, 30 mM MgCl₂) was added to 50 μM final concentration. After 15 min of incubation at 37°C samples were mixed with Laemmli sample buffer and incubated for 3 min at 95°C with vigorous shaking. Proteins were separated on 12% SDS polyacrylamide gel and signal was detected on Medical X-ray Blue/MXBE Film (Carestream).