| Assay Method Information | |
| | ADP-Glo Kinase Assay |
| Description: | The ADP-Glo Kinase Assay Kit was used for the experiments. The following buffer solutions were prepared: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT. The tested compound samples were dissolved in DMSO and added to the screening system at a certain starting concentration of, for example, 10 uM, diluted 3-fold, while setting a DMSO control and an unkinase control. The optimal concentrations of Pd3Kalpha, PI3Kalpha H1047R, substrate (PIIP2) and ATP were prepared with buffers. The enzyme reaction system included: buffer, ATP 25 uM, kinase substrate (PIP2, 50 ug/mL), kinase PI3Kalpha (0.15 ug/mL), PI3Kalpha H1047R (0.05 g/mL), etc. The reaction system was reacted at room temperature for 1 hour, then terminated by adding a terminal reagent (ADP-Glo reagent, 5 uL), and the ADP content in the system was detected using a detection reagent (Kinase Detection Reagent, 10 uL). Signal data was collected using the Envision instrument. Calculated the % inhibition according to the following formula: % inhibition=(DMSO control signal value - sample signal value)! (DMSO control signal value ???unkinase control signal value). |
| Affinity data for this assay | |
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