| Assay Method Information | |
| | Mobility Shift Assay |
| Description: | The MSA (Mobility Shift Assay) method was used to detect the effects of small molecule inhibitors on the kinase activity of EGFR wild type and its mutants (EGFRwt, EGFR-L858R, EGFR-L858R-T790M-C797S, EGFR-L858R-C797S). 10 μL of kinase solution at 2.5 times the final concentration was added to the compound wells and positive control Max wells; 10 μL of 1x kinase buffer was added to the negative control Min wells. The mixture was centrifuged at 1000 rpm for 30 seconds, and the reaction plate was shaken to mix the solution well and then incubated at room temperature for 10 minutes. A mixed solution of ATP and kinase substrate at 5/3 times the final concentration was prepared in 1x kinase buffer. 15 μL of a mixed solution of ATP and substrate at 5/3 times the final concentration was added to initiate the reaction. The 384-well plate was centrifuged at 1000 rpm for 30 seconds,and shaken to mix the solution well and then incubated at room temperature for the corresponding time. 30 μL of stop detection solution was added to stop the kinase reaction, and the mixture was centrifuged at 1000 rpm for 30 seconds, and then to mixed well by shaking. The conversion rate was read using Caliper EZ Reader. |
| Affinity data for this assay | |
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