CHEMBL5737833

KinaseGlo Assay: Kinase phosphorylation reactions were performed in a buffered medium containing 20 mM HEPES pH 7.5 (KOH), 0.1% BSA, 10 mM MgCl2, 1 mM EGTA (pH 7.2), plus or minus 2 mM CaCl2 14. The phosphorylation reaction mixture of 40 μM peptide substrate (Syntide-2, peptide sequence: Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys (SEQ ID NO: 12)) (GenScript, Piscataway, USA), 19.48 nM of 14 TgCDPK1, 90 to 0.0005 μM serial dilutions of inhibitor in a total volume of 25 μl, was initiated by the addition of 10 μM ATP. The reaction was terminated after 30 minutes incubation at 30° C. by addition of excess EGTA (5 mM final concentration). Internal positive and negative controls were included in each assay run.