CHEMBL3887401

Radiometric Protein Kinase Assay: A radiometric protein kinase assay (33PanQinase Activity Assay) was used for measuring the kinase activity of DYRK1A protein kinase. All kinase assays were performed in 96-well FlashPlates from Perkin Elmer (Boston, Mass., USA) in a 50 ul reaction volume. The reaction cocktail was pipetted in four steps in the following order: 20 ul of assay buffer (standard buffer); 5 ul of [gamma-33P]-ATP solution (in H2O); 5 ul of test compound (in 10% DMSO); 10 ul of substrate/10 ul of enzyme solution (premixed).The assay contained 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 uM Na-orthovanadate, 1.2 mM DTT, 50 ug/ml PEG20000, 1 uM [gamma-33P]-ATP (approx. 3x105 cpm per well), protein kinase, and substrate.For the determination of inhibitory profiles, DYRK1A protein kinase was used, which was purchased from Invitrogen Corporation. The kinase was expressed in 519 insect cells as human recombinant GST-fusion protein.