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Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching

Nature Protocols volume 10, pages 660680 (2015) | Download Citation

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Abstract

Proteins within most macromolecular complexes or organelles continuously turn over. This turnover results from association and dissociation reactions that are mediated by each of the protein's functional domains. Thus, studying organelle or macromolecular formation from the bottom up using theoretical and computational modeling approaches will necessitate the determination of all of these reaction rates in vivo. Yet current methods for examining protein dynamics either necessitate highly specialized equipment or limit themselves to basic measurements. In this protocol, we describe a broadly applicable method based on fluorescence recovery after photobleaching (FRAP) for determining how many reaction processes participate in the turnover of any given protein of interest, for characterizing their apparent association and dissociation rates, and for determining their relative importance in the turnover of the overall protein population. Experiments were performed in melanoma M2 cells expressing mutant forms of ezrin that provide a link between the plasma membrane and the cortical actin cytoskeleton. We also describe a general strategy for the identification of the protein domains that mediate each of the identified turnover processes. Our protocol uses widely available laser-scanning confocal microscopes, open-source software, graphing software and common molecular biology techniques. The entire FRAP experiment preparation, data acquisition and analysis require 3–4 d.

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Change history

  • 01 July 2015

     In the version of this article initially published, the files described in the Supplementary Methods were missing and have now been uploaded and made accessible as supplementary information with the published version of the article. The error has been corrected in this file as of 1 July 2015.

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Acknowledgements

The authors acknowledge the University College London (UCL) Comprehensive Biomedical Research Centre for generous funding of microscopy equipment. M.F. was funded by a Human Frontier of Science Program, Young investigator grant to G.C. (RGY 67/2008). G.C. was supported by a University Research Fellowship from the Royal Society.

Author information

Affiliations

  1. The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

    • Marco Fritzsche
  2. London Centre for Nanotechnology and Department of Cell and Developmental Biology, University College London, London, UK.

    • Guillaume Charras

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Contributions

M.F. and G.C. developed the concept and designed the experimental approach. M.F. carried out the experiments and implemented the data analysis. G.C. and M.F. wrote the article.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Marco Fritzsche or Guillaume Charras.

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DOI

https://doi.org/10.1038/nprot.2015.042

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