In vitro kinase assay

One microgram of recombinant substrate was combined with 250 ng of active kinase [PAK1/CDC42 (SignalChem) and 100 µm GTP (Thermo Fisher)] and incubated in kinase buffer [50 mm PO₄ pH 7.4, 150 mm NaCl, 20 mm MgCl₂, 0.1 mg/ml BSA, 1 mm dithiothreitol] with phosphatase inhibitor (Roche), 20 µm cold adenosine tryphosphate (ATP) (Invitrogen) and 1.2 µl of 0.01 mCi/µl ³²P ATP (PerkinElmer) for 1 h at 30°C. The kinase reaction was terminated by the addition of NuPAGE LDS sample buffer and sample reducing agent (Invitorgen) followed by boiling for 15 min. The samples were ran on a NuPAGE 4–12% Bis-Tris Gel (Invitrogen). The gel was coomassie stained (InstantBlue, VWR) for 20 min and exposed to X-ray film (GE) for 1 h.