| Assay Method Information | |
| | Enzymatic Activity Inhibition Assay |
| Description: | Table B: The enzymatic assay used to determine activity was a Luminescence assay using a Microplate Reader ClarioStar Plus. The enzymatic reaction was carried out in assay buffer (40 mM TRIS-HCl pH 7.4-7.6, 20 mM MgCl2, 2.5 mM MnCl2, 0.05 mM DTT, 0.1 mg/ml BSA). The compounds were dispensed on a 384 well Diamond Well Plate (Axigen, Cat #P-384-120SQ-C—S) using the Biomek FX liquid handling system at 80× solutions of compounds in DMSO. 2× Protein-Substrate mix (final concentration 1.5 nM of EGFR (D770_N771insNPG) and 50 ng/μl of Poly(Glu, Tyr)) was prepared in 1× Assay buffer and 4p1 of mixture per well was added into 384w white Reaction plate with NBS (Coming, Cat #4513). 4 μl of Poly(Glu, Tyr) substrate w/o EGFR in 1× buffer was used for negative control. Plates were centrifuged for 1 min at 200 g. Next step the Compounds were added to Reaction plate using Biomek station via following steps: 1 μl of 80× compounds (in DMSO) were mixed thoroughly with 39 μl of 2× 10 μM ATP in Assay Buffer, then 4 μl of this mixture was added to Reaction plate with 4 μl of Protein-Substrate mix. Plates were centrifuged for 1 min at 200 g and incubated for 1 h at rt. Next 4 μL of ADP-Glo reagent (Promega, ADP-Glo™ Kinase Assay, Cat #V9102) per well was added. Plates were incubated for 30 min at rt. Then 8 μL of Kinase detection reagent (Promega, ADP-Glo™ Kinase Assay, Cat #V9102) per well was added and the Luminescence was measured using Microplate Reader. |
| Affinity data for this assay | |
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