A practical guide to single-molecule FRET.
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.
Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, Illinois 61801, USA.
Rahul
R
Roy
Sungchul
S
Hohng
Taekjip
T
Ha
D000465:Algorithms; D001703:Biophysics / Q000295:instrumentation / Q000379:methods*; D001709:Biotechnology / Q000379:methods; D001710:Biotin; D019921:Biotinylation; D002138:Calibration; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D010100:Oxygen / Q000737:chemistry; D011789:Quantum Theory; D012984:Software; D013053:Spectrophotometry / Q000379:methods
D005456:Fluorescent Dyes; D001710:Biotin; D010100:Oxygen