| Assay Method Information | |
| | Enzyme-linked Immunosorbent Assay (ELISA) |
| Description: | The substrate of poly (Glu, Tyr) 4:1 (20 lg/mL) (Sigma, St. Louis, MO, USA) was precoated in 96-well ELISA plates, and the kinase was incubated with the compounds in reaction buffer (50 mM HEPES pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2, 0.2 mM Na3VO4, 1mM DTT) containing 10 µM ATP at 37 °C for an hour. The plates were washed with PBS and incubated with antiphosphotyrosine (PY99) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and horseradish peroxidase-conjugated antibody (Calbiochem, SanDiego, CA, USA) successively. The results were visualized using o-phenylenediamine (OPD) and read on a multiwell spectrophotometer (VERSAmaxTM; Molecular Devices, Sunnyvale, CA, USA). |
| Affinity data for this assay | |
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