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Assay Method Information |
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| | HTRF Assay |
| Description: | Table 4-5: Purified His-tagged TEAD protein (YAP Binding Domain, amino acids 217-447) is pre-incubated with Europium labelled anti-His antibody tracer (Perkin Elmer Cat #AD0110). Small molecule Inhibitors are then incubated with the TEAD-Eu protein complex for 30 minutes or 4 hours to allow for binding to TEAD protein. Biotinylated YAP peptide for TEAD-YAP assays (AA's 50-100) or biotinylated TAZ peptide for TEAD-TAZ assays (AA's 13-57) that has been pre-incubated with streptavidin −xl665 acceptor (CIS-Bio Cat #610SAXAC) or is added to the compound-TEAD mix. The TEAD-YAP-inhibitor mixture is then incubated for 60 minutes at room temperature. All reactions are carried out in a polystyrene plate. After 60 minutes, the plate is read on a plate reader using TR-FRET mode with wavelengths of 665 nm/615 nm. If YAP or TAZ binds to TEAD as expected, a TR-FRET signal results from the proximity of YAP or TAZ and TEAD after binding. If an inhibitor such as peptide 17 (Selleckchem Cat #S8164) interferes with YAP-TEAD or TAZ-TEAD binding, the disruption of the YAP or TAZ:TEAD interaction results in a decrease in TR-FRET signal. The potency of compounds as YAP:TEAD or TAZ:TEAD protein-protein interaction (PPi) inhibitors is determined by an IC50 or EC50 value generated using a non-linear four parameter curve fit. |
| Affinity data for this assay | |
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