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| | In Vitro c-Met Enzyme Assay |
| Description: | The c-Met kinase activity as tested with enzyme-linked immunosorbent assays (ELISAs) as previously reported protocol [Qiang et al., Bioorg. Med. Chem., 24:3353-3358; Shi et al., Bioorg. Med. Chem., 22:4735-4744]. Briefly, reaction cocktail containing c-met kinase and various concentrations of measured compound or 0.1% DMSO (negative control) was incubated at 37 °C for five minutes. Then the mixture of ATP and substrate peptide was added and incubated for another 30 min. Afer that, the reaction was terminated and transferred into a pre-coated 96-well plate, and incubated for another 1 h at 37 °C. Phosphate-buffered saline (PBS) containing 0.1% Tween 20 (T-PBS) was applied to wash the plate three times. anti-phosphotyrosine (PY99) antibody was then added. Horseradish peroxidase-conjugated goat anti-mouse IgG was added after 30 min. Finally, the plate was analyzed with multi-well spectrophotometer at 450 nM. |
| Affinity data for this assay | |
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