| Assay Method Information | |
| | Binding Assay |
| Description: | Binding measurements were performed in parallel sets of either WT/G12D/G12C/G12V KRAS or WT K/H/N RAS proteins in GDP and/or GTPγS-loaded forms.Biacore instrument was desorbed and docked with a Series S Sensor Chip SA. The proteins were diluted to 50 μg/mL with the assay buffer (50 mM HEPES, 150 mM NaCl, 10 μM GDP for GDP-loaded proteins or 10 μM GTPγS for GTPγS-loaded proteins, 5 mM MgCl2, 0.5 mM TCEP, 5% glycerol, 0.02% Tween-20, 2% DMSO, pH 7.2) and immobilized at a flow rate of 3 μL/min at 10° C. with a contact time of 3-10 min. to capture ˜3000-4000 RUs of proteins on the surface. The functionalized surface was then equilibrated with assay buffer for approximately 1 hour. Un-functionalized SA surfaces with no immobilized protein served as reference for binding kinetic analysis. Compound binding kinetics were measured in either multi-cycle or single-cycle kinetic format. |
| Affinity data for this assay | |
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