| Assay Method Information | |
| | PLK4 Biochemical Assay |
| Description: | Activity of human recombinant PLK4 (ThermoFisher, cat #PV6396) was measured by quantification of adenosine diphosphate (ADP) using the ADP-Glo Kinase Assay Kit (Promega, cat #V9102). Test compounds were solubilized in dimethyl sulfoxide (DMSO) and dispensed into 384-well white polystyrene nonbinding plates (Greiner, cat #781094) using the Echo acoustic dispenser (Labcyte Inc.) in a 11-point 3-fold titration in duplicates. 5 µL of 1.0 nM PLK4 protein in assay buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% BSA, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT) was added to the plates. Test compounds and PLK4 were incubated for 15 minutes at room temperature (RT). Then 5 µL of a 16 µM adenosine triphosphate (ATP) (Promega, cat #V915B) and 9.3 µM Myelin Basic Protein (MBP) (SignalChem, cat #M42-51N) substrate solution in assay buffer was added and the reaction mixture was incubated for 6 hours at RT. The final concentration of PLK4, ATP and MBP in the reactions were 0.5 nM, 8.0 µM and 4.7 µM, respectively. Reactions were stopped and the remaining ATP depleted by adding 10 µL of ADP-Glo reagent (Promega, cat#V912B) and incubating for 40 minutes at RT. The simultaneous conversion of the remaining ADP to ATP and measurement of the newly synthesized ATP was achieved by addition of 20 µL Kinase Detection reagent (Promega, cat #V914B), incubation for 30 min at RT, and luminescence detection using the EnVision plate reader (PerkinElmer). Reactions lacking PLK4 were used as 100% inhibition controls. Reactions containing DMSO alone were used as 0% inhibition controls. The IC50 values reported in Table 2 were determined using four parameter non-linear regression curve fit. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |