In vitro Phosphorylation Assay Protocol

In vitro phosphorylation assays were performed as previously described^{46 (link)}. In brief, cells from 100 µL of the E. coli cultures that were started for protein expression were collected by centrifugation and then resuspended in 100 µL of SDS sample buffer (200 mM Tris-HCl, 8% SDS, 40% glycerol, 400 mM DTT, and 0.2% bromophenol blue), followed by boiling for 10 min. Hereafter, the samples were centrifuged for 2 min at maximum speed in an Eppendorf centrifuge, and 8 µL of the supernatant were loaded onto a precast mini-PROTEIN TGX Polyacrylamide Gel (BIORAD). After running for around 100 min at 160 V, the gel was incubated in fixation solution (50% methanol, 10% acetic acid in H₂O), overnight. Next, the gel was washed in deionized water for 30 min twice, and the phosphorylated proteins were stained using a Pro-Q Diamond solution (Invitrogen). Subsequently, the staining solution was removed by washing the gel in de-staining solution (20% acetonitrile, 50 mM sodium acetate), and proteins were stained with CBB.