| Assay Method Information | |
| | Phosphoinositide 3-kinase α (PI3Kα) Biochemical Assay |
| Description: | A solution was prepared containing 0.2 nM Anti-HIS-Terbium (Cisbio, 64CUSTAZU), 3.7 nM designed-probe and 1.0 nM His-PIK3CA/PIK3R1 (p110 alpha/p85 alpha) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest for liquid handling, the detection antibody/enzyme/probe solution (2 uL per well) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at room temperature for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls. |
| Affinity data for this assay | |
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