Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells

Erik L. Snapp1, Ramanujan S. Hegde2

1 Albert Einstein College of Medicine, Bronx, New York, 2 NICHD, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.9
DOI:  10.1002/0471143030.cb1709s32
Online Posting Date:  October, 2006
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Fluorescence resonance energy transfer (FRET) refers to the nonradiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor). Measurement of FRET between two fluorophore‐labeled proteins can be used to infer the subnanometer spatial and temporal characteristics of protein interactions in their native cellular environment. Multiple experimental methods exist for measuring FRET. The method that can be most widely and simply implemented, quantified, and interpreted is the acceptor‐photobleaching FRET technique. In this method, the presence of FRET between a donor and acceptor is revealed upon destruction (by photobleaching) of the acceptor. Acceptor photobleaching can be exploited to detect changes in the composition and organization of subunit proteins within a multiprotein complex and to even gain insight into relative stoichiometries of proteins within the complex. In this unit, strategies, tools, and background for designing and interpreting acceptor‐photobleaching FRET experiments in cells are described.

Keywords: Cy3; Cy5; photobleach; stoichiometry; FRET

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Table of Contents

  • Background Information
  • Optimization of Acceptor‐Photobleaching FRET
  • Interpretation of Results
  • Reciprocal FRET
  • Automated Image Analysis
  • Conclusion
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

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