| Assay Method Information | |
| | Kinase Assay |
| Description: | The kinase activity was measured by using the commercial ADP Hunter Plus assay available from DiscoveRx (#33-016), which is a homogeneous assay to measure the accumulation of ADP, a universal product of kinase activity. The enzyme, PI3K (p110alpha /p85alpha was purchased from Carna Biosciences (#07CBS-0402A). The assay was done following the manufacturer recommendations with slight modifications: Mainly the kinase buffer was replace by 50 mM HEPES, pH 7.5, 3 mM MgCl2, 100 mM NaCl, 1 mM EGTA, 0.04% CHAPS, 2 mM TCEP and 0.01 mg/ml BGG. The PI3K was assayed in a titration experiment to determine the optimal protein concentration for the inhibition assay. To calculate the IC50 of the ETP-compounds, serial 1:5 dilutions of the compounds were added to the enzyme at a fixed concentration (2.5 ug/mL. The enzyme was preincubated with the inhibitor and 30 uM PIP2 substrate (P9763, Sigma) for 5 min and then ATP was added to a final 50 uM concentration. |
| Affinity data for this assay | |
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