| Assay Method Information | |
| | EGFR Inhibition Assay |
| Description: | Compounds 1-32 were dissolved in 100% DMSO and diluted to the appropriate concentrations with 25 mM HEPES at pH 7.4. In each well, 10 μL of compound was incubated with 10 μL (12.5 ng for HER-2 or 5 ng for EGFR) of recombinant enzyme (1:80 dilution in 100 mM HEPES) for 10 min at room temperature. Then, 10 μL of 5 mM buffer (containing 20 mM HEPES, 2 mM MnCl2, 100 μM Na3VO4, and 1 mM DTT) and 20 μL of 0.1 mM ATP-50 mM MgCl2 was added for 1 h. Positive and negative controls were included in each plate by incubation of enzyme with or without ATP-MgCl2. At the end of the incubation period, the liquid was aspirated, and plates were washed three times with wash buffer. A 75 μL (400 ng) sample of europium labeled anti-phosphotyrosine antibody was added to each well for a further 1 h of incubation. After washing, enhancement solution was added and the signal was detected by Victor (Wallac, Massachusetts, USA) with excitation at 340 nm and emission at 615 nm. |
| Affinity data for this assay | |
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