| Assay Method Information | |
| | Nucleotide Exchange Assay (NEA)-HTRF Assay for KRas G12C |
| Description: | The effects of compounds on SOS1 catalyzed displacement of GDP by GTP on KRas proteins was examined by homogeneous time-resolved fluorescence (HTRF). 30 μM 6×his labeled KRas G12C recombinant protein and 80 μM fluorochrome DY647 labeled GDP were co-incubated in a labeling buffer (1 mM DTT, 7.5 mM EDTA, 25 mM Tris-HCl, 45 mM NaCl) at 20° C. away from light for 2 hours. Protein quantification was performed after purification on the NAP-5 column to determine the concentration of KRas G12C-GDP.1000× stock solutions of compounds at a concentration gradient of 3.16 were prepared by using DMSO, and diluted 250 times by a reaction buffer (40 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 10 mM MgCl2, 1 mM DTT, 0.002% Triton X-100) into 4× stock solutions of compounds. KRas G12C-GDP/Tb working solution (40 nM KRas G12C-GDP, 1×anti-his Tb) and SOS1/GTP working solution (0.2 μM SOS1, 200 μM GTP) were prepared by using the reaction buffer.5 μl of 4×stock solution of a compound and 10 μl of KRas G12C-GDP/T working solution were added to each well of 96-well plate with white unclear bottom, and 5 μl of reaction buffer was added to the well of control group in stead of the 4×stock solution of a compound. After incubation at 20° C. away from light for 15 minutes, 5 μl of SOS1/GTP working solution was added and incubated at 20° C. away from light for 2 hours, and then a value of fluorescence intensity was read (excitation wavelength: 320 nm, and emission wavelengths: 615 nm and 665 nm). Besides, group TO was established with 10 μl of the reaction buffer and 10 μl of the KRas G12C-GDP/Tb working solution, and the value of fluorescence intensity was directly read. |
| Affinity data for this assay | |
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