| Assay Method Information | |
| | HIPK2 Enzymatic Activity Inhibition |
| Description: | The half maximal inhibitory concentration (IC50) with respect to HIPK2 inhibition was determined for the compounds of the invention by using Z′-LYTE from ThermoFisher Scientific's SelectScreen Biochemical Kinase Profiling Service (Waltham, MA). Compounds were screened in 1% DMSO (final) in the well. For 10 point titrations, 3-fold serial dilutions were conducted from the starting concentration. The 2 HIPK2/Ser/Thr 09 mixture was prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. The black 384-well plate (Corning Cat. #4514) were used for the assay.100 nL of 100 test compound in 100% DMSO, 2.4 uL of kinase buffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA), 5 uL of 2 HIPK2/Ser/Thr 09 mixture, and 2.5 uL of 4 ATP solution were mixed and agitated for 30 seconds. Then the Kinase Reaction was incubated at room temperature for 60 minutes. The final 10 uL Kinase Reaction consisted of 3.53-19 ng HIPK2 and 2 μM Ser/Thr 09 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 uL of a 1:512 dilution of Development Reagent A is added. After 30 second of plate shake, the reaction plate was incubated for another 60 min at room temperature. Then the plate was read on a fluorescence plate reader (CLARIOstar, BMG Labtech, Germany) as plate reader and data was analyzed using XLfit from IDBS. The dose response curve was curve fit to model number 205 (sigmoidal dose-response model). If the bottom of the curve does not fit between −20% & 20% inhibition, it was set to 0% inhibition. If the top of the curve does not fit between 70% and 130% inhibition, it was set to 100% inhibition. |
| Affinity data for this assay | |
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