| Assay Method Information | |
| | Kinase Inhibition Assay |
| Description: | Mobility shift assays were performed to determine the compound's inhibitory activity against EGFR delta19del/T790M/C797S, EGFR WT, and IGF1R kinases. The enzyme reaction scheme is as follows: 1. Prepare 1*kinase buffer as follows. Final 1* Kinase buffer concentration HEPES PH 7.5 (mM) 50 Brij-35 0.0150% DTT (mM) 2 Mgcl2, Mncl2 (mM) 10 2. Preparation of compound concentration gradient: The starting concentration of the test compound is 3000 nM or 100 nM, dilute it in the 384-source plate to a 100% DMSO solution of 100 times the final concentration, and use precision to dilute the compound 3 times to 10 concentrations. Use the Dispenser Echo 550 to transfer 250 nL of 100x the final concentration of compound to the destination plate OptiPlate-384F. 3. Prepare a kinase solution with 2.5 times the final concentration using 1x Kinase buffer. 4. Add 10 uL of 2.5 times the final concentration of kinase solution to the compound wells and positive control wells respectively; add 10 uL of 1x Kinase buffer to the negative control wells. 5. Centrifuge at 1000 rpm for 30 seconds, shake and mix the reaction plate and incubate at room temperature for 10 minutes. 6. Use 1x Kinase buffer to prepare a mixed solution of ATP and Kinase substrate at 5/3 times the final concentration. 7. Add 15 uL of a mixed solution of ATP and substrate at 5/3 times the final concentration to start the reaction. 8. Centrifuge the 384-well plate at 1000 rpm for 30 seconds, mix well by shaking, and incubate at room temperature for the corresponding time. 9. Add 30 uL of stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and shake to mix. 10. Use Caliper EZ Reader to read conversion rates. 11. Calculation formula |
| Affinity data for this assay | |
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