In vitro kinase assay of EGFR protein and inhibitors

The recombinant proteins of the kinase domain of wild-type EGFR and EGFR-C797S/T790M/L858R were purchased from Signal Chem. The inhibitors were purchased as described earlier. The appropriate amount of target proteins calculated on the basis of the ADP-Glo assay manufacturer's protocol was incubated in 96-well half area white plates with serially diluted inhibitor over a 10-dose range from 0.0002, nM to 10 μM for 10 min at room temperature. ATP at concentration of 1, 10, 100 and 1,000 nM was mixed with 100 μg ml⁻¹ substrate and added to a kinase protein–inhibitor mixture, and then incubated for 60 min at room temperature. After the kinase reaction, an equal volume of ADP-Glo Reagent was added to terminate the kinase reaction, and the remaining ATP was depleted. The Kinase Detection Reagent was added both to convert ADP to ATP and to allow the newly synthesized ATP to be measured using the luciferase/luciferin reaction. The light generated was measured using a luminometer.