Confocal single-molecule FRET for protein conformational dynamics

Yan Wen Tan, Jeffrey A. Hanson, Jhih-Wei Chu, Haw Yang

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

14 Scopus citations

Abstract

Single-molecule FÓ§rster-type resonance energy transfer (smFRET) is a unique technique capable of following conformational motions of individual protein molecules. The direct observation of individual proteins provides rich information that would be washed away in ensemble measurements, hence opening up new avenues for establishing the protein structure-function relationships through dynamics. Retrieving dynamics information of biomolecular motions via smFRET, though, requires careful experiment design and rigorous treatment of single-molecule statistics. Here, we describe the rudimentary steps for an smFRET experiment, including sample preparation for the microscope, building of critical parts for single-molecule FRET detection, and a robust methodology for photon-by-photon data analysis.

Original languageEnglish
Title of host publicationProtein Dynamics
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages51-62
Number of pages12
ISBN (Print)9781627036573
DOIs
StatePublished - 1 Jan 2014

Publication series

NameMethods in Molecular Biology
Volume1084
ISSN (Print)1064-3745

Keywords

  • Model free
  • Poisson statistics
  • Protein immobilization
  • Single photon counting

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