In vitro protein kinase assay

In all, 21-µL reaction solutions were prepared containing 200 ng of protein kinase and 2 µg of substrate protein in 1× kinase assay buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 10 mM magnesium acetate, 0.1% (v/v) 2-mercaptoethanol and 0.1 mM [γ³²P]-ATP). Reactions were conducted at 30 °C for 30 min at 1050 rpm and terminated via the addition of 7 µL of NuPAGE 4× LDS sample buffer containing 8% (v/v) 2-mercaptoethanol. For in vitro kinase assays involving the use of small-molecule inhibitors, reaction solutions containing all the required components were incubated at 30 °C for 10 min at 1050 rpm prior to the addition of 0.1 mM [γ³² P]-ATP. Reactions were then performed as detailed previously. Samples were incubated at 95 °C for 5 min, and subsequently centrifuged at 5.0 × 10³ × g for 1 min. Samples were loaded onto NuPAGE 4–12% Bis-Tris precast polyacrylamide gels and resolved via SDS-PAGE. Polyacrylamide gels were subsequently stained with InstantBlue Coomassie Protein Stain (Expedeon) to visualise the resolved recombinant proteins, and imaged using the ChemiDoc Imaging System (Bio-Rad). ³²P radioactivity was analysed via autoradiography using Amersham Hyperfilm (GE Healthcare Life Sciences).