| Assay Method Information | |
| | CK2 Alpha 1 Enzymatic Activity Inhibition |
| Description: | The half maximal inhibitory concentration (IC50) with respect to CK2 alpha 1 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 CSNK2A1 (CK2 alpha 1)/Ser/Thr 11 mixture is 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 CSNK2A1 (CK2 alpha 1)/Ser/Thr 11 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 0.68-25.7 ng CSNK2A1 (CK2 alpha 1) and 2 μM Ser/Thr 11 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 1:16 dilution of Development Reagent B is added. After 30 second of plate shake, the reaction plate was incubated for another 60 min at room temperature. |
| Affinity data for this assay | |
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