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| | EGFR Tyrosine Kinase Activity Assay |
| Description: | Kinase assays were performed in 96-well plates (Multiscreen Durapore, Millipore) using [γ-32P]ATP (Amersham Biosciences) and the synthetic polymer poly(Glu4/Tyr)(Sigma Chemicals) as a phosphoacceptor substrate. Tested compounds were dissolved in 1H-NMR DMSO, and the final concentration of 1H-NMR DMSO in assay solutions was0.1% (total kinase activity) or 0.01% (EGFR kinase activity), which was shown to have no effect on kinase activity. 20 ng of EGFR (purified from human carcinoma A431 cells; Sigma Chemicals) was incubated for 1 h at 28°C using various concentrations of tested compounds in kinase buffer (HEPES 50 mM pH 7.5, bovine serum albumin (BSA) 0.1 mg/mL, MnCl2 10 mM, MgCl2 5 mM, Na3VO4 100 μM, dithiothreitol (DTT) 0.5 mM, poly(Glu4/Tyr) 250 μg/mL, ATP 5 μM, [γ-32P]ATP 0.5 μCi). |
| Affinity data for this assay | |
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