| Assay Method Information | |
| | Inhibitory Activity for EGFR (L858R/T790M/C797S) Kinase |
| Description: | A 50 ng/μL EGFR (L858R/T790M/C797S, BPS) stock solution was diluted with a kinase buffer (50 mM HEPES, 10 mM MgCl2, 2 mM DTT, 1 mM EGTA, 0.01% Tween 20), and 6 μL of 1.67× working solution at 0.00167 ng/μL (final concentration: 0.001 ng/μL) was added to each well. Different compounds dissolved in DMSO were added to wells using a nanoliter pipettor, resulting in the final concentrations of the compounds of 10 nM to 0.0024 nM (4-fold gradient, 7 concentrations in total), and blank control wells (without enzyme) and negative control wells (with enzyme, plus vehicle DMSO) were set, 2 replicate wells for each concentration. After enzyme was reacted with the compound or vehicle for 30 min, 5×ATP at 25 μM (final concentration: 5 μM, Sigma) prepared with a kinase buffer was mixed with 5× substrate at 0.5 μM (final concentration: 0.1 μM, ULight-poly GT, PerkinElmer) at a ratio of 1:1 (v:v), and the mixture was added to each well at 4 μL/well. After being sealed with a film, the plate was incubated at room temperature for 2 h, and then 5 μL of 4×EDTA at 40 mM (final concentration: 10 mM) was added to each well. After the plate was incubated for 5 min at room temperature, 5 μL of 4× detection reagent at 8 nM (final concentration: 2 nM, Eu-anti-phospho-tyrosine antibody, PerkinElmer) was added to each well. After being incubated at room temperature for 1 h, the plate was read using a PerkinElmer Envision multi-mode microplate reader (excitation: 320 nm, emission: 665 nm), and IC50 was calculated by four-parameter fitting. |
| Affinity data for this assay | |
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