| Assay Method Information | |
| | Inhibitory Effect of Compound I on the Enzymatic Activity of PI3Kδ and PI3Kγ In Vitro |
| Description: | 1) Preparation of buffer salt solution: A 10× buffer salt solution with a pH of 7.5 containing 500 mM HEPES, 500 mM NaCl, and 30 mM MgCl2 was prepared with ultrapure water and stored at 4° C. for later use. Before use, the above buffer salt solution was diluted to a 3.33× buffer salt solution and BSA was added to a final concentration of 0.333 mg/mL.2) A 100× reference compound (compound I) was prepared with a starting concentration of 100 nM, diluted in a 3-fold serial dilution to 10 concentrations and transferred to the corresponding 384 microwell plate at 50 nL/well. In the control group, 50 nL/well of DMSO was added.3) 3.33×PI3K solutions were prepared with the 3.33× buffer salt solution, PI3Kδ at a final concentration of 0.25 nM, PI3Kγ at a final concentration of 0.4 nM. A 3.33×PIP2:3PS solution was prepared, mixed with the enzyme solutions at a volume ratio of 1:1, and added to the 384 microwell plate in 3 μL/well. The mixed solution of buffer salt solution/PIP2:3PS was added in the complete inhibition control group. The mixture was mixed well, centrifuged, and incubated for 20 minutes at 23° C.4) The 384 microwell plate was removed, and 2.5×ATP solutions, at a final concentration of 40 μM (PI3Kδ) and 25 μM (PI3Kγ) respectively, were prepared with ultrapure water, added to the 384 microwell plate in 2 μL/well, mixed well, centrifuged, and incubated for 120 minutes at 23° C. Then, 5 L/well of ADP-Glo reagent was added, mixed well, centrifuged, and incubated for 60 minutes at 23° C. 10 μL/well of kinase detection reagent was added, mixed well, centrifuged, and incubated for 30 minutes at 23° C. The luminescence value was read using Envision.4. Experimental results:The experiment used the ADP-Glo chemiluminescence method as the enzymatic activity detection method to determine the inhibitory effect of the test compound I on the enzymatic activity of PI3Kδ and PI3Kγ. |
| Affinity data for this assay | |
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