| Assay Method Information | |
| | In Vitro Ras-Raf Binding Inhibitory Activity Assay |
| Description: | The in vitro Ras-Raf binding inhibitory activity of the compound of the present invention was evaluated by an ELISA method described below. GST-c-Raf-1 RBD diluted with a Ras-Raf binding buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, and 1% Triton-X100) was added to each well of a glutathione-coated 96-well plate (Thermo Fisher Scientific.), and was incubated at 30° C. for 1 hour, to immobilize Raf on the well. The unbound free Raf was eliminated by washing the well with the Ras-Raf binding buffer three times. Subsequently, GTPTS-bound HRasG12V diluted with the Ras-Raf binding buffer and each compound solution (final DMSO concentration: 10%) were applied to each well, and the resulting mixture was then incubated at 30° C. for 1 hour, so that Ras and Raf were allowed to bind to each other. Thereafter, the plate was washed with the Ras-Raf binding buffer twice, and was then subjected to a blocking treatment at room temperature for 20 minutes, using TBS-Tween {10 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.05% (w/v) Tween-20}-5% (w/v) bovine serum albumin (BSA). After that, an anti-HRas antibody (C-20, Santa Cruz) or an anti-HRas antibody (259, Santa Cruz), which had been 1000 times diluted with TBS-Tween-5% BSA, was added to the reaction mixture, and incubated at room temperature for 1 hours. After the treatment with the primary antibody, the plate was washed with TBS-Tween-5% BSA three times, and a horseradish peroxidase-labeled secondary antibody against rabbit immunoglobulin G (GE Healthcare) of 1000 times diluted with TBS-Tween-5% BSA, was added to the plate, followed by incubation at room temperature for 1 hour. After the treatment with the secondary antibody, the plate was washed with TBS-Tween-5% BSA three times, and a substrate solution (TMB) was then added to the plate, followed by incubation at room temperature for 15 minutes for coloration. Finally, 2 M H2SO4 was added to stop color development (coloration reaction kit: Nacalai Tesque). |
| Affinity data for this assay | |
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