| Assay Method Information | |
| | Disrupting KRAS-PI3Kα Interaction by SPR Inhibition Assay |
| Description: | SPR binding experiments were collected on a Biacore 8K Instrument (Cytiva). Neutravidin (Pierce) was amine coupled to the carboxymethylated dextran surface of a CM5 sensor chip (Cytiva) using standard amine coupling chemistry. The CM5 chip surface was first activated with 0.1 M N-hydroxy succinimide and 0.4 M N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide at a flow rate of 20 μL/min using 20 mM HEPES pH 7.4, 150 mM NaCl as the running buffer. Next, neutravidin was diluted to 20 μg/mL in 10 mM sodium acetate (pH 4.5) and injected on all flow cells until a density of approximately 10,000 response units (RU) was immobilized. Activated amine groups were quenched with an injection of 1 M ethanolamine (pH 8.0). 300-500 RU of avi-tagged full length PI3Kα was captured on all flow cells in 20 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP, 0.05% tween 20, 5% DMSO buffer. KRAS-Q25A at 20 μM was mixed with 8 concentrations of compound (50 nM-100 μM) and injected over the full-length PI3Kα at 30 μL/min and 25° C. Steady-state levels of KRAS binding were recorded and fit with a 4-parameter inhibition model to determine the IC50 values. |
| Affinity data for this assay | |
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