| Assay Method Information | |
| | EGFR Biochemical Assay |
| Description: | Biochemical activity was determined using a chelation-enhanced fluorescence assay. EGFR enzyme was incubated with DMSO-dissolved inhibitors in reaction buffer (50 mM HEPES pH 7.4, 10 mM MgCl2, 1% glycerol, 0.0125% Brij-35, 1.2 mM DTT, and 0.02% BSA) for 30 min at RT before initiating reactions with AssayQuant peptide AQT0734 (10 μM) and ATP (see enzyme and ATP details in table below). Reaction times were 360 min (EGFR WT), or 150 min (EGFR (d746-750) T790M C797S; EGFR L8585R T790M C797S). Total substrate phosphorylation was measured as fluorescence intensity (excitation 360 nm, emission 480 nm) on a PerkinElmer Ensight plate reader. IC50 values were calculated by fitting background-subtracted fluorescence values to a log(inhibitor) vs. response Hill equation.Final enzyme ATPCat concentration, concentration,Protein Supplier No. nM nMEGFR WT SignalChem E10- 20 25112G EGFR SignalChem E10- 2.5 100(d746-750) 122UG T790M C797S EGFR SignalChem E10- 1.25 50(L858R 122VG T790M C797S)[1772] |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |