| Assay Method Information | |
| | Biochemical Assay |
| Description: | Biochemical Assays for Inhibition of Wild-Type and Mutant EGFRs.The inhibitory activities against EGFR WT, EGFR Δ752-759 mutant, EGFR Δ746-750/T790M mutant, EGFR Δ746-750/C797S mutant, EGFR Δ746-750/T790M/C797S mutant. EGFR L858R mutant, EGFR L858R/T790M mutant, EGFR L858R/C797S mutant, and EGFR L858R/T790M/C797S mutant were evaluated at Reaction Biology Corporation (See, www.reactionbiology.com) using HotSpot assay platfrom, a radiometric assay based on conventional filter-binding assays, that directly measures kinase catalytic activity toward a specific substrate (Anastassiadis T, et al. Comprehensive Assay of Kinase Catalytic Activity Reveals Features of Kinase Inhibitor Selectivity. Nat Biotechnol. 2011, 29:1039-45). Briefly, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer; 20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO. Compounds were delivered into the reaction, followed 20 minutes later by addition of a mixture of ATP (Sigma, St. Louis MO) and 33P ATP (Perkin Elmer, Waltham MA) to a final concentration of 10 μM. Reactions were carried out at room temperature for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman Inc., Piscataway, NJ). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data was expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. |
| Affinity data for this assay | |
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