Labeling proteins for single-molecule FRET

Cold Spring Harb Protoc. 2012 Sep 1;2012(9):1009-12. doi: 10.1101/pdb.prot071035.


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.

MeSH terms

  • DNA Helicases / chemistry
  • DNA Helicases / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism
  • Fluorescence
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / metabolism
  • Proteins / chemistry*
  • Proteins / metabolism
  • Staining and Labeling / methods*


  • Escherichia coli Proteins
  • Fluorescent Dyes
  • Proteins
  • rep protein, E coli
  • DNA Helicases