| Assay Method Information | |
| | Binding Activity Assay |
| Description: | a) BI-2852 was used as a positive control, with its stock solution as the first dilution point, followed by a 3-fold dilution, making a total of 10+0 dilutions. Similarly, the first dilution point of the test compounds was also their respective stock solutions, followed by a 3-fold dilution, making a total of 11+0 dilutions. Using Echo, 0.2 μL of the gradient-diluted compound solutions were transferred into a 384-well plate, with each compound tested in duplicate. The final DMSO concentration was 1%. The plate was centrifuged at 1000 rpm for 1 minute. The final concentrations of the positive controls were 100, 33.33, 11.11, 3.70, 1.23, 0.412, 0.137, 0.046, 0.015, 0.005, and 0 μM. The final concentrations of the test compounds were 200, 66.67, 22.22, 7.41, 2.47, 0.27, 0.091, 0.03, 0.0152, 0.01, and 0 μM.b) KRAS_WT from the kit was prepared with GTP at a final concentration of 10 μM in the dilution buffer, and 5 μL was transferred into the 384-well reaction plate. The plate was centrifuged at 1000 rpm for 1 minute.c) Subsequently, 5 μL of the SOS1 mixture was transferred into the 384-well reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated at 25° C. for 15 minutes.d) 10 μL of the detection mixture was transferred into the 384-well reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated overnight at 4° C.e) The excitation wavelength at 665 nm and emission wavelength at 615 nm were read using an Envision multimode plate reader. The 665/615 ratio signal intensity was used to characterize the enzyme activity.f) The raw data was analyzed. |
| Affinity data for this assay | |
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