| Assay Method Information | |
| | Inhibitory Activity on KRAS G12D Nucleotide (GDP-GTP) Exchange Reaction |
| Description: | For the measurement of the inhibitory activity of compounds on GDP-GTP exchange rate of recombinant KRAS G12D, BODIPY FL GDP-bound KRAS G12D protein was incubated with various concentrations of compound in a reaction buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 2 mM DTT, 0.1% Tween 20) at 25° C. for 1 hour. After the incubation, recombinant SOS1 and GMPPNP (guanosine-5′-[((β, γ)-imido]triphosphate, tetralithium salt) (Jena Bioscience GmbH, Jena, Germany) were added and incubated at room temperature for 30 minutes to proceed SOS1-dependent GDP-GTP exchange reaction on KRAS G12D. Replacement of BODIPY FL GDP by GMPPNP was measured by calculating the ratio of fluorescence intensities of BODIPY FL before and after the exchange reaction. |
| Affinity data for this assay | |
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