Journal
NATURE METHODS
Volume 5, Issue 6, Pages 507-516Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.1208
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Funding
- Howard Hughes Medical Institute Funding Source: Medline
- NIGMS NIH HHS [R01 GM065367] Funding Source: Medline
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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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