Abstract
Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique for measuring distances in the 30-80 Å range in which excitation energy of a donor molecule is transferred to an acceptor via interaction between two induced dipoles. A variant of smFRET is based on total internal reflection (TIR) microscopy. This protocol describes the labeling of protein for smFRET with TIR microscopy. It is based on a labeling procedure for E. coli Rep helicase. A different assay (e.g., different chemical conditions) may be required for other proteins.
Original language | English |
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Pages (from-to) | 1009-1012 |
Number of pages | 4 |
Journal | Cold Spring Harbor Protocols |
Volume | 7 |
Issue number | 9 |
DOIs | |
State | Published - Sep 2012 |