Review Article | Published:

A practical guide to single-molecule FRET

Nature Methods volume 5, pages 507516 (2008) | Download Citation

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Abstract

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

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Acknowledgements

We acknowledge I. Rasnik, S. Mckinney, C. Joo, R. Clegg, S. Myong, members of Ha group and K. Drexhage for expert advice and discussion; S. Syed (University of Illinois) for procurement of the dyes and reagents; and P. Cornish, M. Brenner and L. Supriya for carefully reading the manuscript. C. Joo prepared the video instruction on PEG slide preparation. Authors' work on single-molecule FRET was funded by the US National Institutes of Health, National Science Foundation career award and Howard Hughes Medical Institute. S.H. was also supported by Research Settlement Fund for the new faculty at Seoul National University (Korea), Ministry of Science and Technology grant (RH0-2005-000-01003-0, 2007) and Basic Science Research Grant from the Korea Research Foundation.

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Affiliations

  1. Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, Illinois 61801, USA

    • Rahul Roy
    •  & Taekjip Ha
  2. Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, Illinois 61801, USA.

    • Rahul Roy
    •  & Taekjip Ha
  3. Department of Physics and Astronomy, Seoul National University, San 56-1 Sillim 9-dong, Gwanak-gu, Seoul 151-747, Korea.

    • Sungchul Hohng
  4. Howard Hughes Medical Institute, 1110 West Green Street, Urbana, Illinois 61801, USA.

    • Taekjip Ha

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Correspondence to Taekjip Ha.

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https://doi.org/10.1038/nmeth.1208

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