| Assay Method Information | |
| | Inhibition of PI3Kdelta Efficacy Assay |
| Description: | The ADP-Glo™ kinase assay was used to assess the inhibition of PI3Kδ with our compounds. The utilized proteins and assay reagents were obtained from the ADP-Glo™ Kinase Assay Kit (Promega). Initially, a 2 mM stock solution of the test compound (dissolved in DMSO) was serially diluted in five-fold concentrations using DMSO to obtain eight working solutions 1 (200×) Subsequently, each of the eight working solutions 1 was further diluted in a 20-fold gradient by adding 5 μL into 95 μL of ddH2O, resulting in eight working solutions 2 (10×). In a 384-well white flat-bottom plate, 2 μL of 2.5×PI3Kδ kinase solution and 1 μL of working solution 2 (10×) were added to each well and mixed, followed by a 15-minute incubation at room temperature (RT). Subsequently, 2 μL of 2.5×PI and ATP solution mixture were added to each well, mixed, and incubated at RT for 60 minutes. Then, 5 μL of ADP-Glo (containing 10 mM MgCl2) reagent was added to each well, mixed, and incubated at RT for 40 minutes. Finally, 10 μL of the Kinase Detection Reagent was added to each well, mixed, and incubated at RT for 40 minutes. Luminescence values were recorded using a multi-mode microplate reader with luminescence channel settings, and the inhibitory rate was calculated using the following formula:%Inhibitionrate=[(Negative-testcompound)/(Negative-Blank)]*100Data were analyzed using GraphPad Prism with log(inhibitor) vs. response—Variable Slope(four parameters) fitting calculations for IC50 values. |
| Affinity data for this assay | |
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