In vitro kinase assay

0.2 μg of purified GST-Oct4 proteins were used in cold in vitro kinase assays with purified recombinant kinases. All kinases were purchased from Proqinase (Germany). Kinase reactions were performed in kinase buffer (60mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 0.5mM ATP) for 30 min at 30°C. Then reactions were stopped by the addition of 5X SDS-PAGE loading buffer and assessed by Westernblot.

For radioactive in vitro kinase assay GST-Oct4 was incubated with Aurkb in kinase buffer (60 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 0.25 mM ATP) with 0.1 µM γ-³²P-ATP (NEG002A250UC, purchased from PerkinElmer, Waltham, Massachusetts) for 30 min at 30°C. Reactions were then stopped by the addition of 5X SDS-PAGE loading buffer and loaded for separation on 8% SDS-PAGE gel. After staining with Coomassie Blue, the gels were dried and exposed to films.