A practical guide to single-molecule FRET.

Nature methods (2008-05-31)
Rahul Roy, Sungchul Hohng, Taekjip Ha
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 550, for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 550 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Sigma-Aldrich
Atto 550-Biotin, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 550 azide, BioReagent, suitable for fluorescence, ≥80.0% (HPCE)
Sigma-Aldrich
Atto 550 amine, BioReagent, suitable for fluorescence

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