| Assay Method Information | |
| | KRAS(GMPPNP)-RAF1 HTRF Binding Assay |
| Description: | The test compounds were prepared as lOmM stock solution in 100% DMSO. The stock solution was then serially diluted 3 -fold in 100% DMSO to 10 concentrations. 200nL of each diluted compound solution was transferred to 384-well plate in duplicate. To each well, 10 pL of enzyme solution containing Biotin-KRAS G12D or WT protein with final concentration of 5 nM or 2 nM in the assay buffer (50mM HEPES, pH7.5, 50mM NaCl, 5mM MgCl2, ImM DTT, 0.1% BSA, 0.01% Triton X-100), respectively. For negative control, 10 pL of assay buffer was added instead. The plate was incubated at room temperature for 20 min. 5 pL of GST-RAF1 (final concentration at 8 nM) was subsequently added to each well. Then 5 pL of lx anti-GST-XL665 and lx Streptavidin-Tb were added for detection. After one-hour incubation at room temperature, all samples were subjected to read the TR-FRET signal on Envision with excitation at 340nm and emission fluorescence at 615 nm and 665 nm. IC50 values were then calculated by plotting dose-response curves and then using the XLfit application in Excel software. |
| Affinity data for this assay | |
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