| Assay Method Information | |
| | Kinase Inhibition Assay |
| Description: | WT EGFR and EGFR[L858R/T790M] kinase detection: In 5× kinase buffer A, WT EGFR or EGFR[L858R/T790M] kinase was mixed with different concentrations of compounds prepared by pre-dilution for 10 minutes. Each concentration was tested in duplicate. The corresponding substrate and ATP were added, and reaction was performed at room temperature for 20 minutes (negative and positive controls were provided: the negative control was a blank control, and the positive control was erlotinib). After completion of the reaction, detection reagents (reagents in the HTRF Kinase TK kit) were added. After incubating for 30 minutes at room temperature, enzyme activity in the presence of various concentrations of the compounds of the present disclosure was determined by Evnvision microplate reader, and inhibitory activity of different concentrations of the compounds on enzyme activity was calculated. The inhibitory activity of different concentrations of the compounds on enzyme activity was then fitted by Graphpad 5.0 software according to the four-parameter equation, and the IC50 value was calculated. |
| Affinity data for this assay | |
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