| Assay Method Information | |
| | Test of Inhibitory Activity of the Compound of the Present Invention on PI3K Kinase |
| Description: | The experimental procedure is briefly described as follows: the test compound was first dissolved in DMSO to prepare a stock solution, and then the buffer was prepared according to the buffer formulation provided in the reagent manual (HEPES 50 mM, MgCl2 3 mM, EGTA 1 mM, CHAPS 0.03%, NaCl 100 mM, pH7.5). The buffer was used for gradient dilution. The final concentration of the test compound in the reaction system ranged from 1000 nM to 0.05 nM. The ATP Km values of PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ were determined by using a gradient diluted ATP solution (from ADP-Glo™ Kinase Assay Kit), and the ATP concentration in the reaction system was set to 10 μM based on the ATP Km value obtained in the experiment. The reaction was carried out in a 384-well microplate. First, the compound and a certain amount of PI3Kα, PI3Kβ, PI3Kγ or PI3Kδ protein were added to the wells, and incubated at room temperature for 15 minutes. Then ATP solution and PIP2:3PS (the final concentration was 0.01 mg/mL) were added to the reaction solution, and incubated at room temperature with shaking for 60 minutes. Then 5 μL of ADP-Glo Reagent (containing 10 mM MgCl2) was added to the reaction system, and continued to incubate with shaking for 40 minutes at room temperature. Then 104, Kinase Detection Reagent was added to the reaction system, and continued to incubate with shaking for 40 minutes at room temperature. After incubation, the chemiluminescence intensity value of each well was measured in Luminescence mode on the microplate reader. By comparing with the luminous intensity ratio of control group (0.1% DMSO), the percentage inhibition rate of the compound at each concentration was calculated. The GraphPad Prism 5 software was used to perform nonlinear regression analysis on the compound concentration logarithmic value vs. the inhibition rate to obtain IC50 values of the compounds, and the result was shown on Table 1. |
| Affinity data for this assay | |
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