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Assay Method Information

Assay Name:  n/a
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 (p110α/p85α was purchased from Carna Biosciences (#07CBS-0402A). The assay was done following the manufacturer recommendations with slight modifications, mainly that 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 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. The reaction was carried out for 1 hour at 25° C. Reagent A and B were sequentially added to the wells and plates were incubated for 30 min at 37° C. Fluorescence counts were read in a Victor instrument (Perkin Elmer) with the recommended settings (544 and 580 nm as excitation and emission wavelengths, respectively). Values were normalized against the control activity included for each enzyme (i.e., 100% PI3 kinase activity, without compound). These values were plotted against the inhibitor concentration and were fit to a sigmoid dose - response curve by using the model sigmoidal Four-Parameter Logistc implement for Activity base - software.
Affinity data for this assay
 

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Last update November 1, 2007
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