Imaging and identifying impurities in single-molecule FRET studies

Chirlmin Joo, Taekjip Ha

Research output: Contribution to journalArticle

Abstract

Single-molecule (sm) 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. To study the conformational changes of individual molecules over extended time periods, the molecules must be immobilized on a coated surface that allows highly specific tethering of DNA, RNA, or protein. Nevertheless, there are always some fluorescent impurities on the surface and, without experience in sm imaging, it is often difficult to distinguish molecules of interest from impurities. This protocol describes the use of an imaging buffer that enhances the photostability of cyanine dyes used for smFRET, with emphasis on distinguishing molecules of interest from impurities.

Original languageEnglish (US)
Pages (from-to)1109-1112
Number of pages4
JournalCold Spring Harbor Protocols
Volume7
Issue number10
DOIs
Publication statusPublished - Oct 2012
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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