Labeling proteins for single-molecule FRET
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Updated: May 18, 2026

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level
Published on: April 19, 2019
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.
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