| Assay Method Information | |
| | EGFR Activity Inhibition Assay |
| Description: | In the case of Km ATP, EGFR T790M/L858R protein kinase activity was tested by Caliper mobility shift assay (referring to J Biomol Screen 14:31, 2009).Assay method: the compound to be tested was dissolved in DMSO and then diluted with a kinase buffer solution (50 mM HEPES-pH7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT). 5 μl of 10% DMSO with 5-fold final reaction concentration of the compound dissolved was added into a 384-well plate, a control well without the compound was added 5 μl of 10% DMSO, and a control well without kinase activity was added 5 μl of the kinase buffer solution. After adding 10 μl of 2.5-fold diluted EGFR (Cama, Cat. No 08-115, Lot. 13-CBS-0005M) kinase solution, the incubation was performed for 10 minutes at room temperature, and 10 μl of 2.5-fold diluted substrate solution Peptide FAM-P22 (GL Biochem, Cat. No. 112393, Lot. No. P130408-ZB112393) was further added. After being incubated for 60 minutes at 28° C., 25 μl of stopping solution (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 m MEDTA) was added to terminate the reaction. The conversion ratio data was read on Caliper EZ Reader II (Caliper Life Sciences). The conversion ratio was converted to the inhibition ratio data.A curve was plotted with the concentration of the compound and the inhibition ratio as the abscissa and ordinate values. XLFit excel add-in version 4.3.1 software was used to fit the curve and calculate IC50. Inhibition ratio %=(max-conversion ratio)/(max-min)×100, wherein max refers to the conversion ratio of the control well in which the compound is absent in DMSO and min refers to the conversion ratio of the control well without kinase activity. |
| Affinity data for this assay | |
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