| Assay Method Information | |
| | PI3Kα and PI3Kδ Biochemical Assay |
| Description: | PI3Kα and PI3Kδ Kinase reactions were performed in a 5 μL volume in low-volume 384-well plates. Typically, PerkinElmer model 6008280 plates were used. The 1× kinase reaction buffer consisted of 50 mM HEPES pH 7.5, 3 mM MgCl2, 0.03% CHAPS, 1 mM EGTA, 100 mM NaCl, and 2 mM DTT. PI3Kα (ThermoFisher, #PV4788; the final PI3Kα concentration: 120 ng/mL) or PI3Kδ enzyme solution (ThermoFisher, #PV6451; the final PI3Kδ concentration: 250 ng/mL) was added to the plate, compounds were serially diluted to the final maximal concentration of 100 nM (3 fold series dilution, a total of 10 doses), and the PIP2:3PS (the final concentration: 10 μg/mL) and ATP solution (the final ATP concentration: 10 μM) was added to the 384-well plate. The plate was incubated at 25° C. for 1 hour. ADP-Glo reagent buffer (5 μL) was added to each well. The plate was sealed and incubated for 40 min at 25° C. ADP-Glo detection buffer (10 μL) was added to each well, and the plate was incubated for 40 min at 25° C. and read on a plate reader configured for Luminescence. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |