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2015 ; 1278
(ä): 545-53
Nephropedia Template TP
Methods Mol Biol
2015[]; 1278
(ä): 545-53
PMID25859975
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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) ?
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.
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