CHEMBL5729936

In Vitro Kinase Assay: In vitro kinase analysis was performed by using HTScan EGF Receptor Kinase Assay Kit (#7909) and HTScan HER2/ErbB2 Kinase Assay Kit (#7058) from Cell Signaling Technology Company. Operation steps refer to the specification of the used kits, and the method was used to measure the inhibition effects of the compound to be tested on substrate peptide phosphorylation of EGFR or Her2 receptor tyrosine kinase. At room temperature, ATP and substrate peptide as well as the compound to be tested were incubated in kinase reaction buffer, after a period of incubation, a stop buffer was added to terminate the reaction and the sample was transferred to a streptavidin-coated 96-well plate, the plate was washed and the phosphorylation level of substrate peptide was detected by using HRP-marked antibody against substrate phosphorylation, colorated with TMB, terminated the reaction with 2M sulfuric acid. Absorption at 450 nm wavelength was detected, and IC50 value (μM) was calculated.