title: Estrogen receptor alpha/co-activator interaction assay: TR-FRET.
abstract: Time-resolved fluorescence resonance energy transfer, TR-FRET, is a time-gated fluorescence intensity measurement which defines the relative proximity of two biomolecules (e.g., proteins, peptides, or DNA) based on the extent of non-radiative energy transfer between two fluorophores with overlapping emission/excitation spectra. In these assays, an excited lanthanide ion acts as a "donor" that transfers energy to an "acceptor" fluorophore through dipole-dipole interactions. A FRET signal is reported as the ratio of acceptor to donor emission following donor excitation. When a donor-conjugated protein interacts with an acceptor-conjugated protein, the donor and acceptor fluorophores are brought in close proximity allowing energy transfer from the donor to the acceptor resulting in a FRET signal. Because the lanthanide donors have a long emission half-life, the energy transfer measurement can be time-gated, which dramatically reduces assay interference (due to background autofluorescence and direct acceptor excitation) and thereby increases data quality. Here, we describe a TR-FRET assay that monitors the interaction of the estrogen receptor (ER) α ligand binding domain (labeled with a terbium chelate via a streptavidin-biotin interaction) with a sequence of coactivator protein SRC3 (labeled directly with fluorescein) and the disruption of this interaction with a peptide and a small molecule inhibitor.
authors: Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL, 61801, USA.
authors: Terry W
authors: TW
authors: Moore
authors: Jillian R
authors: JR
authors: Gunther
authors: John A
authors: JA
authors: Katzenellenbogen
mesh_terms: D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008024:Ligands; D056921:Nuclear Receptor Coactivator 3 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D015398:Signal Transduction
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D008024:Ligands; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3