title: Labeling proteins for single-molecule FRET.
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.
authors.forename: Chirlmin
authors.initials: C
authors.lastname: Joo
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011506:Proteins; C506744:rep protein, E coli; D004265:DNA Helicases