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Homo-FRET Imaging as a Tool to Quantify Protein and Lipid Clustering

Authors

  • Dr. Arjen N. Bader,

    1. Department of Molecular Biophysics, Universiteit Utrecht, Princetonplein 1, 3584 CC Utrecht (The Netherlands), Fax: (+31) 30 253  2706
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  • Dr. Sandra Hoetzl,

    1. Department of Membrane Enzymology, Universiteit Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands)
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  • Dr. Erik G. Hofman,

    1. Department of Cellular Dynamics, Universiteit Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands)
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  • Jarno Voortman,

    1. Department of Cellular Dynamics, Universiteit Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands)
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  • Dr. Paul M. P. van Bergen en Henegouwen,

    1. Department of Cellular Dynamics, Universiteit Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands)
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  • Prof. Dr. Gerrit van Meer,

    1. Department of Membrane Enzymology, Universiteit Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands)
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  • Prof. Dr. Hans C. Gerritsen

    Corresponding author
    1. Department of Molecular Biophysics, Universiteit Utrecht, Princetonplein 1, 3584 CC Utrecht (The Netherlands), Fax: (+31) 30 253  2706
    • Department of Molecular Biophysics, Universiteit Utrecht, Princetonplein 1, 3584 CC Utrecht (The Netherlands), Fax: (+31) 30 253  2706
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  • FRET: Förster Resonance Energy Transfer

Abstract

Homo-FRET, Förster resonance energy transfer between identical fluorophores, can be conveniently measured by observing its effect on the fluorescence anisotropy. This review aims to summarize the possibilities of fluorescence anisotropy imaging techniques to investigate clustering of identical proteins and lipids. Homo-FRET imaging has the ability to determine distances between fluorophores. In addition it can be employed to quantify cluster sizes as well as cluster size distributions. The interpretation of homo-FRET signals is complicated by the fact that both the mutual orientations of the fluorophores and the number of fluorophores per cluster affect the fluorescence anisotropy in a similar way. The properties of the fluorescence probes are very important. Taking these properties into account is critical for the correct interpretation of homo-FRET signals in protein- and lipid-clustering studies. This is be exemplified by studies on the clustering of the lipid raft markers GPI and K-ras, as well as for EGF receptor clustering in the plasma membrane.

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