| Assay Method Information | |
| | Inhibitory activity on EGFR |
| Description: | The Assay Buffer was prepared, and the composition of the Assay Buffer was 50 mM Hepes (Gibco, 15630), 10 mM MgCl2, 2 mM DTT, 1 mM EGTA, and 0.010% Tween 20. EGFR kinase (Carna, 08-115), ATP (Sigma, A7699), and ULight-poly GT (PE, TRF0100-M) working solutions were prepared using the Assay Buffer. EDTA and Eu-labeled anti-phosphotyrosine (PT66) antibody (PE, AD0069) working solutions were prepared using the Detection Buffer. 6 μL of EGFR kinase at the corresponding concentration (final concentration: 0.006 ng/μL) was added to the compound groups and the control group, while 6 μL of the Assay Buffer was added to the blank group, and then the compounds (with the concentration set to 1000 nM at the maximum, 4-fold dilution, 7 concentration gradients) were added using a nanoliter pipettor. The mixture was incubated at room temperature for 30 min, and then 4 μL of the mixture of ATP (final concentration: 5 μM) and ULight-poly GT (final concentration: 100 nM) was added. After incubation at room temperature for 2 h, 5 μL of EDTA (final concentration: 10 mM) was added to stop the reaction, and finally 5 μL of antibody (final concentration: 2 nM) was added. After incubation at room temperature for 1 h, signal values were detected at 665/615 nM, the four-parameter analysis was performed, the dose-response curves were fit, and IC50 was calculated. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |