Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors (original) (raw)

Nature Methods volume 5, pages 401–403 (2008)Cite this article

Abstract

Fluorescence resonance energy transfer (FRET) with fluorescent proteins is a powerful method for detection of protein-protein interactions, enzyme activities and small molecules in the intracellular milieu. Aided by a new violet-excitable yellow-fluorescing variant of Aequorea victoria GFP, we developed dual FRET–based caspase-3 biosensors. Owing to their distinct excitation profiles, each FRET biosensor can be ratiometrically imaged in the presence of the other.

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Figure 1: Caspase-3 biosensors based on dual FRET pairs.

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Figure 2: Live-cell imaging with dual FRET pairs.

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Figure 3: Control experiments with noncleavable FRET constructs.

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Acknowledgements

This work was funded by the University of Alberta, the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council of Canada and Alberta Ingenuity. We thank A.M. Sierra and I.S. Goping for technical assistance and helpful discussion. R.E.C. holds a Canada Research Chair in Bioanalytical Chemistry.

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Authors and Affiliations

  1. Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, T6G 2G2, Alberta, Canada
    Hui-wang Ai & Robert E Campbell
  2. National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, 32310, Florida, USA
    Kristin L Hazelwood & Michael W Davidson

Authors

  1. Hui-wang Ai
  2. Kristin L Hazelwood
  3. Michael W Davidson
  4. Robert E Campbell

Contributions

H.A. contributed to the conceptual development, performed experiments, analyzed data and assisted in manuscript preparation; K.L.H. performed photobleaching experiments and imaging of fusion proteins; M.W.D. contributed to experimental design, analyzed photobleaching data and assisted with manuscript preparation; and R.E.C. contributed to conceptual development, data analysis and writing of the manuscript.

Corresponding author

Correspondence toRobert E Campbell.

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Competing interests

R.E.C. and H.A. are listed as authors on a US patent application describing mTFP1.

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Ai, Hw., Hazelwood, K., Davidson, M. et al. Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors.Nat Methods 5, 401–403 (2008). https://doi.org/10.1038/nmeth.1207

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