| Assay Method Information | |
| | In Vitro EphB4 Enzyme Inhibition Assay |
| Description: | This assay detects inhibitors of EphB4-mediated phosphorylation of a polypeptide substrate using Alphascreen luminescence detection technology. The recombinant EphB4 was incubated with biotin-poly-GAT in presence of magnesium- ATP. The reaction was stopped by addition of EDTA, together with streptavidin-coated donor beads which bind the biotin-substrate containing any phosphorylated tyrosine residues. Anti-phosphotyrosine antibodies present on acceptor beads bind to phosphorylated substrate, thus bringing the donor and acceptor beads into close proximity. Subsequent excitation of the donor beads at 680nm generated singlet oxygen species that interact with a chemiluminescer on the acceptor beads, leading to light emission at 520-620nm. The signal intensity is directly proportional to the level of substrate phosphorylation and thus inhibition is measured by a decrease in signal. The resulting assay signal was determined on the Perkin Elmer EnVision plate reader. The minimum value was subtracted from all values, and the signal plotted against compound concentration to generate IC50 data. |
| Affinity data for this assay | |
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