| Assay Method Information | |
| | Inhibitory Activity of the Compounds of the Invention Against ROS1, NTRK and ALK and their Drug-Resistant Kinases |
| Description: | Inhibition of protein kinase activity by compounds was carried out on the Radio-tagged HotSpot kinase experimental platform of Reaction Biology Corporation. Fresh reaction solution (20 mM TIEPESpH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 100 DMS0) containing corresponding substrates was prepared, cofactor and kinase to be tested were added into the above solution and mixed gently. Echo550 pipetting system was used to add the test compound DMSO solution to each well (the blank control group was added with the corresponding volume of DMSO), then 33P-ATP (with a final specific activity of 0.01 μCi/μL) was added to start the reaction. The reaction solution was incubated at room temperature for 120 minutes. Transferred the incubated reaction solution to P81 ion exchange chromatographic paper (Whatman #3698-915), eluted with 0.7500 phosphoric acid solution, and the amount of radioactive phosphorylated substrate remaining on the chromatographic paper was detected. |
| Affinity data for this assay | |
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