| Assay Method Information | |
| | PI3K Biochemical Assay |
| Description: | PI3K Biochemical PotencyPI3Kα (p110α/p85a), PIK3C δ, PIK3Cβ (p110β), PIK3Cγ (pp110γ) kinase reaction solutions of PI3Kα (Invitrogen, Cat. No. PV4788), PIK3Cδ (Millipore, Cat. No. 14-604-M), PIK3Cβ (Eurofins, Cat. No. 14-603-K), PIK3Cγ (Invitrogen, Cat. No. PR8641C) enzymes were prepared in 1× kinase buffer at 4-fold of the final concentration (final concentration: PI3Kα 0.7 nM, PIK3C δ 3 nM, PIK3Cβ 4.8 nM, PIK3Cγ 11 nM) of each reagent in the assay. 2.5 μl of kinase solution was added to each well of the 384-well assay plate, which contains 2.5 μl of compounds with serially diluted concentration. 2× substrate solution was prepared with PIP2 substrate and ATP in 1× kinase reaction buffer at 2-fold of the final concentration of each reagent in the assay. 5 μl of substrate solution was added to each well of the assay plate to start reaction. The assay plate was incubated at room temperature for 1 hour. 5 μl reaction mix was transferred to a new 384 well plate. 5 μl of ADP-Glo reagent (Promega, Cat. No. v9102/3) was added to each well of the new assay plate to stop the reaction. The plate was shaken slowly and equilibrated for 40 minutes. 10 μl kinase detection reagents were added to each well, which was equilibrated for 60 minutes before read on a plate reader (Envision) for luminescence. |
| Affinity data for this assay | |
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