| Assay Method Information | |
| | FRET Assay for REV-ERB Ligands |
| Description: | The capacity for compounds to function as ligands for either REV-ERBα or REV-ERBβ was assessed using a fluorescent resonance energy transfer assay that detects the interaction between these receptors and the Nuclear Receptor Corepressor (NCoR) protein (the ID2 corepressor interaction domain peptides is used). This interaction is known to be ligand dependent and thus this assay that can detect an alteration of the affinity of these two proteins is able to detect ligands. The His-tagged ligand binding domain (LBD) of either REV-ERBα or REV-ERBβ and fluorescein labeled NCoR ID2 peptide (Life Technologies #PV4624) is used in these assays. The His-tagged LBDs were expressed in E. coli (amino acids 281-614 REV-ERBα and 381-579 REV-ERBβ) and labeled with a Terbium (Tb) labeled anti-His antibody (Life Technologies #PV5895). The assay buffer was PBS (phosphate buffered saline) with 5 mM DTT (dithiothreitol). The final concentration of various reagents in the assay are: REV-ERB LBD (either isoform) [5 nM], Fluorescein labeled NCoR ID2 peptide [250 nM], Tb labeled anti-his antibody [10 nM], dimethylsulfoxide [1%] and test compound [varying concentrations]. The assay was performed in Corning NBS black 384-well plates in a total volume of 20 μl. The assay plate was incubated for 1 hour at room temperature protected from light and then TR-FRET was assessed on a Biotek plate reader with the following excitation/emission pairs (340 nm/495 nm and 340 nm/520 nm. |
| Affinity data for this assay | |
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