Preparing sample chambers 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 in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. A variant of smFRET is based on total internal reflection (TIR) microscopy, which can be set up in two ways, either using an oil-immersion (objective-type) or a water-immersion (prism-type) lens. To study the conformational changes of individual molecules over extended time periods, molecules must be localized in space. This protocol describes the preparation of sample chambers with either bovine serum albumin (BSA)- or polyethylene glycol (PEG)-coated slides to which single molecules can be tethered for use in FRET studies.

Original languageEnglish (US)
Pages (from-to)1104-1108
Number of pages5
JournalCold Spring Harbor protocols
Volume7
Issue number10
DOIs
StatePublished - Oct 1 2012

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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