| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | The composition of the assay buffer used for the activity measurement was as follows: 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 7.5 mM MgCl2, 3 mM KCl, 0.01% Tween 20, 0.1% BSA, and 1 mM DTT. Herein, an enzyme reaction was performed using a peptide substrate labeled with ATP at the concentration of 1 mM and biotin at the concentration of 0.5 μM. The analysis of the EGFR activity inhibitory effect of the compound was performed according to the following analytical reaction recipe.Component 1: 4 μl of EGFR mutant enzymeComponent 2: 2 μl of compound solutionComponent 3: 4 μl of ATP and biotin-labeled peptide mixtureThe enzyme reaction was initiated by mixing the component 1 and the component 2 first and then adding the component 3 thereto. After reacting the mixture at 37° C. for 2 hours, 10 μl of a measurement solution consisting of streptavidin-XL665 and europium-labeled anti-phosphotyrosine antibody provided by Cisbio was added to the enzyme reaction solution, followed by reaction at room temperature for 1 hour. Finally, the ratio of the fluorescence values at 615 nm and 665 nm was calculated using Envision equipment of Perkin-Elmer to quantitatively measure the enzyme activity and confirm the inhibitory ability of the compound. The values measured at 7 concentrations of the compound were analyzed using Prism program (version 5.01, Graphpad Software, Inc.), and the IC50 value, an index of the inhibitory ability of the compound, was calculated. |
| Affinity data for this assay | |
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