For in vitro enzyme kinetic and inhibition studies, wild type or mutant EGFR kinases (residues 696-1022 of the human EGFR) were expressed and purified as GST-fusion proteins in Sf9 insect cells essentially as described55 . Kinetic parameters were determined using the ATP/NADH coupled assay system in a 96-well plate, as described previously 55 . The reaction mixture contained 0.5 mg/mL BSA, 2 mM MnCl2, 1 mM PEP (2-(Phosphonooxy)-2-propenoic acid, Sigma-Aldrich, Cat. P-7002), 1 mM TCEP, 0.1 M HEPES (pH7.5), 2.5 mM tyrosine kinase substrate poly[E4Y] (P7244, Sigma-Aldrich), 1/50 of the final reaction mixture volume of PK/LDH enzyme (Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle, Cat. P-0294, Sigma-Aldrich), 0.5 mM NADH and 0.1~0.5 μM EGFR (0.1 μM for L858R, 0.5 μM for WT and 0.2 μM for T790M/V948R). 1 mM ATP was added last to start the reactions. Steady state initial velocity data were drawn from the slopes of the A340 curves and fit to the Michaelis-Menten equation to determine Vm and Km values. EGFR inhibition by Mig6 was measured using the same assay and Kiapp values were determined by fitting inhibition curves to the Morrison equation using Graphpad PRISM software.
Poly e4y
Manufactured by Merck Group
Sourced in United States
Poly[E4Y] is a synthetic polymer designed for use in various laboratory applications. It serves as a key component in specialized equipment and instruments. The core function of Poly[E4Y] is to facilitate specific chemical and biological processes within controlled environments. No further details can be provided while maintaining an unbiased and purely factual approach.
Automatically generated - may contain errors
3 protocols using poly e4y
1
Kinetic and Inhibition Assay for EGFR
2
Kinase Inhibition Profiling of MT-SYK-03
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The activity of MT-SYK-03 against cSrc kinase and selectivity profile towards other 341 kinases was determined at Reaction Biology Corporation (Malvern, PA, USA) using the standard kinase assay protocol. The reaction mixture containing 0.1 mg/mL of substrate polyE4Y (Sigma, USA), 6xHis-SYK (0.1—0.7 pM), the studied compound MT-SYK-03 or reference R406 (500 nM) and 33P ATP (up to 10 μM, final specific activity 0.225–0.360 μCi) was incubated (120 min) and spotted onto ion exchange filter; unbound phosphate was removed by extensive washing of filters in phosphoric acid. Kinase activity data was expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions based on the radioactivity of reaction products (see Table S2 ). Experiment was replicated twice. IC50 values for kinase listed in Table S3 (having less than 50% residual activity) were measured in 10-dose mode with threefold serial dilution starting at 10 μM and curve fits were obtained using Prism4 Software (GraphPad). Dose response curve of inhibitory activity of MT-SYK-03 against cSrc kinase is presented in Fig. S1 b.
3
Kinetic and Inhibition Assay for EGFR
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