Nature Methods
- 5, 507 - 516 (2008)
Published online: 29 May 2008; | doi:10.1038/nmeth.1208
A practical guide to single-molecule FRETRahul Roy1, 2, Sungchul Hohng3 & Taekjip Ha1, 2, 41
Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, Illinois 61801, USA
2
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, Illinois 61801, USA. 3
Department of Physics and Astronomy, Seoul National University, San 56-1 Sillim 9-dong, Gwanak-gu, Seoul 151-747, Korea. 4
Howard Hughes Medical Institute, 1110 West Green Street, Urbana, Illinois 61801, USA.
Correspondence should be addressed to Taekjip Ha tjha@uiuc.edu 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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