| Assay Method Information | |
| | SOS-Catalyzed Nucleotide Exchange Assay (version 1) |
| Description: | Recombinant KRAS G12C (amino acids 1-169, SEQ ID NO:1), KRAS G12D (amino acids 1-169, SEQ ID NO:2), KRAS G12V (amino acids 1-169, SEQ ID NO:3) and SOS1 (amino acids 564-1049, SEQ ID NO:4) proteins were expressed in E. coli and purified by affinity chromatography. To prepare each BODIPY FL GDP-bound KRAS mutant protein, 50 uM KRAS mutant proteins were incubated with 0.5 mM BODIPY FL GDP (Invitrogen, G22360) in a loading buffer (20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT and 2.5 mM EDTA) for 1 hour on ice. After the incubation, MgCl2 was added to a final concentration of 10 mM, followed by incubation at room temperature for 30 minutes. The mixtures were allowed to pass through a NAP-5 column to remove free nucleotides and purified BODIPY FL GDP-bound KRAS G12C, G12D and G12V proteins were used for compound evaluation. For the measurement of the inhibitory activity of compounds on GDP-GTP exchange rate of recombinant KRAS mutants, each BODIPY FL GDP-bound KRAS mutant protein whose concentration is 25 nM 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 (Jena Bioscience GmbH, NU-401) were added and incubated at room temperature for 30 minutes to proceed SOS1-dependent GDP-GTP exchange reaction on KRAS mutants. |
| Affinity data for this assay | |
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