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Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors


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.
Figure 2: Live-cell imaging with dual FRET pairs.
Figure 3: Control experiments with noncleavable FRET constructs.

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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



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 to Robert 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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–2, Supplementary Methods, Supplementary Notes 1–2 (PDF 4718 kb)

Supplementary Movie 1

Time-lapse movie of the cell shown in Figure 2. (MOV 983 kb)

Supplementary Movie 2

Time-lapse movie of the cell shown in Supplementary Figure 3. (MOV 1120 kb)

Supplementary Movie 3

Time-lapse movie of the cell shown in Supplementary Figure 4. (MOV 1473 kb)

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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).

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