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Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox
Published on: July 5, 2021
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Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox
Mahmoud A S Abdelhamid1, Alice V Rhind-Tutt1, Benjamin Ambrose1
1Sheffield Institute for Nucleic Acids, Department of Chemistry, University of Sheffield.
Journal of Visualized Experiments : Jove
|July 19, 2021
Summary
The new smfBox instrument offers a cost-effective way to perform single-molecule Förster Resonance Energy Transfer (smFRET) measurements on freely diffusing biomolecules. This protocol details its use for precise FRET efficiency measurements in DNA, enhancing accessibility for researchers.
Area of Science:
- Biophysics
- Molecular Biology
- Analytical Chemistry
Background:
- Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique for studying biomolecular dynamics.
- Existing smFRET instruments can be expensive and complex, limiting their widespread adoption.
- There is a need for accessible and standardized methods for smFRET measurements.
Purpose of the Study:
- To present a detailed protocol for using the cost-effective, open-source smfBox instrument.
- To enable precise FRET efficiency measurements in duplex DNA samples.
- To facilitate the democratization of smFRET technology for the broader scientific community.
Main Methods:
- Step-by-step protocol for smfBox instrument operation.
- Detailed procedures for sample preparation and instrument setup/alignment.
- Comprehensive data acquisition and analysis routines, including correction factor determination.
Main Results:
- Demonstration of precise FRET efficiency measurements in duplex DNA.
- Establishment of a standardized approach for smFRET data analysis.
- Validation of the smfBox as a reliable and accessible smFRET measurement tool.
Conclusions:
- The smfBox instrument and accompanying protocol significantly lower the barrier to entry for smFRET studies.
- This work contributes to standardizing smFRET methodologies and analysis.
- The protocol is adaptable to various biomolecular systems, promoting wider use of smFRET.
