| Assay Method Information | |
| | Biochemical Assay Against EGFR L858R/C797S Protein (EGFR LR/CS) |
| Description: | The inhibitory activity of the compounds disclosed herein were measured in a biochemical assay utilizing a recombinant human EGFR L858R/C797S double mutant protein, utilizing detection of chelation-enhanced fluorescence using the sulfonamido-oxine (Sox) chromophore covalently attached to an optimized kinase substrate.The following materials were used: EGFR L858R C797S (Carna Biosciences, Cat #08-563); phosphosens peptide substrate (AssayQuant, Cat #AQT0794); adenosine triphosphate (ThermoFisher, Cat #R0441); assay Buffer: 50 mM HEPES, pH=7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% BSA, 0.01% Brij-35, 1 mM DTT; 384-well Polypropylene Plates (Greiner Cat #781201); 384-well Black Low Volume Plates (Greiner Cat #784076); TopSeal-A Plus (PerkinElmer Cat #6050185); Dimethyl Sulfoxide (Sigma, Cat #D2650); 1 M HEPES (VWR Chemicals, Cat #J848); 2M Magnesium Chloride (Quality Biological, Cat #340-034-721); 0.5M Ethylene Glycol Tetra Acetic Acid (Alfa Aesar, Cat #J60767); 30% Bovine Albumin, Fraction V (Alfa Aesar, Cat #165569); 10% Brij-35, (EMD Millipore, Cat #203728); 1M Dithiothreitol, (Invitrogen, Cat #P2325)In Row A of a 384-well polypropylene plate, was added 48 μL of DMSO. In wells A1 and A24, were added 12 μL of DMSO. Column 1 (16 replicates) served as enzyme+DMSO total signal (0% inhibition). Column 24 (16 replicates) served as no enzyme+DMSO background (100% inhibition). To a well in Row A were added 12 μL of each 10 mM test compound stock (100% DMSO). The master compound plate was transferred to the Bravo and run the “PPAR FRET cmpd serial dilution and intermediate dilution.pro” protocol to generate 16-point serial dilutions (3.16× or 0.5 log dilutions) of each compound. The final intermediate dilution plate contained 4×16-point compound serial dilutions in assay buffer. The DMSO concentration was 10%. The intermediate compound dilution plate was transferred to the Bravo and the “5 uL dispense into ARP.pro” protocol was run. To the black low volume assay ready plates, were added 5 μL of 40 uM Phosphosens peptide sensor/4 mM ATP working solution to each well. The plates were spun for 1 minute at 1200 rpm in an Eppendorf 5810R table-top centrifuge. Next, 10 μl of 2.0 nM EGFR L858R/C797S working solution was added to all wells in columns 1-23. To column 24, was added 10 μL of assay buffer. The final DMSO concentration was 2.5%. The plates were spun for 1 minute at 1200 rpm in an Eppendorf 5810R table-top centrifuge. |
| Affinity data for this assay | |
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