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| | Luminescence Kinase Assay |
| Description: | The assay was performed using Kinase-Glo Plus luminescence kinase assay kit (Promega). The compounds were diluted in 10% DMSO and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. All of the enzymatic reactions were conducted at 30° C. for 25 minutes. The 50 μl reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT, 0.2 mg/ml Poly (Glu, Tyr) substrate, 10 μM ATP and EGFR (Table 2.3.1). After the enzymatic reaction, 50 μl of Kinase-Glo Plus Luminescence kinase assay solution (Promega) was added to each reaction and incubate the plate for 5 minutes at room temperature. Luminescence signal was measured using a BioTek Synergy 2 microplate reader. EGFR activity assays were performed in duplicate at each concentration. |
| Affinity data for this assay | |
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