| Assay Method Information | |
| | KRAS(G12C) and SOS1 Binding Assay |
| Description: | 1× buffer preparation (ready-to-use): Hepes: 5 mM; NaCl: 150 mM; EDTA: 10 mM; Igepal: 0.0025%; KF: 100 mM; DTT: 1 mM; BSA: 005%;The test compounds were 5-fold diluted with DMSO using a multi-channel pipette to the 8th concentration, i.e., diluted from 1 mM to 0.064 μM.The test compound was diluted by gradient with 1× buffer to a working solution with 2% DMSO. 5 μL/well of the working solution was added to corresponding wells, and the corresponding concentration gradient was 20 μM to 0.00128 nM. Duplicate experiment was set. The working solution was centrifuged at 1000 rpm for 1 minute.A mixed working solution of KRAS(G12C) (200 nM) and Mab Anti GST-Eu cryptate (1 ng/μL) was prepared with 1× buffer, and the mixed working solution was incubated at 25° C. for 5 minutes, and 2.5 μL/well of the mixed working solution was added to the corresponding wells.A mixed working solution of SOS1 (80 nM) and Mab Anti 6HIS-XL665 (8 g/μL) was prepared with 1× buffer, and 2.5 μL/well of the mixed working solution was added to the corresponding wells. 2.5 μL of Mab Anti 6HIS-XL665 (8 g/μL) diluent was added to blank wells. At this time, the final concentration gradient of the compound was diluted from 10 μM to 0.64 nM, KRAS(G12C) (500 nM), MAb Anti GST-Eu cryptate (0.25 ng/μL), SOS1 (20 nM), Mab Anti 6HIS-XL665 (2 g/μL), the reaction system was placed at 25° C. for 60 minutes. After the reaction was completed, the multimode microplate reader was used to read HTRF. |
| Affinity data for this assay | |
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