| Assay Method Information | |
| | Competition Binding Assay |
| Description: | Table 3: The Equilibrium dissociation constant reflected the test compound's kinase binding (Kd). The KINOMEscan, a site-directed competition binding assay, was used to measure interactions between test compounds and specific kinases. The kinase-tagged T7 phage strains were derived from the BL21 strain's E. coli host. T7 phage was used to infect E. coli, which was then incubated until lysis occurred. The remaining kinases were produced in HEK-293 cells and tagged with DNA. To create affinity resins for kinase, streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands. The assay consists of three components: a DNA-tagged kinase, an immobilized ligand, and the test compound. An immobilized ligand could compete with the compound binding the kinase active site. The affinity beads were washed with wash buffer and re-suspended in elution buffer following reactions in a polypropylene 384-well plate. qPCR of the kinase-DNA tag was used to assess the test compound's ability to bind with the selected kinase. The assay was carried out in accordance with the manufacturer's instructions (Eurofins DiscoverX Corporation). |
| Affinity data for this assay | |
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