Labeling proteins for single-molecule FRET

Chirlmin Joo, Taekjip Ha

Research output: Contribution to journalArticlepeer-review

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 languageEnglish (US)
Pages (from-to)1009-1012
Number of pages4
JournalCold Spring Harbor Protocols
Volume7
Issue number9
DOIs
StatePublished - Sep 2012
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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