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Analysis of Protein Dynamics with Tandem Fluorescent Protein Timers

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Exocytosis and Endocytosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1174))

Abstract

Fluorescent timers (FTs) are fluorescent proteins that change color with time. FTs can be used as tags to follow protein dynamics in living cells. Recently we described a novel class of FTs called tandem fluorescent protein timers (tFTs). Each tFT is a tandem fusion of two different conventional fluorescent proteins having distinct kinetics of fluorophore maturation. tFTs suitable for studying protein dynamics on different scales can be generated from a broad range of commonly used fluorescent proteins. Here we describe how to establish new tFTs and consider potential pitfalls. We detail a protocol for quantitative fluorescence microscopy imaging and analysis of intracellular protein dynamics with tFTs in the budding yeast Saccharomyces cerevisiae.

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Acknowledgements

We thank Daniel Kirrmaier and Matthias Meurer for help with the experiments and Joseph D. Barry and Leopold Parts for comments and acknowledge support from the European Molecular Biology Organization to AK (EMBO ALTF 1124-2010) and from the Deutsche Forschungsgemeinschaft (SFB1036).

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Authors and Affiliations

  1. Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, Heidelberg, 69120, Germany

    Anton Khmelinskii & Michael Knop

Authors
  1. Anton Khmelinskii
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  2. Michael Knop
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Corresponding author

Correspondence to Michael Knop .

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Editors and Affiliations

  1. Department of Human and Molecular Genetics, VCU Institute of Molecular Medicine, Virginia Commonwealth University, Richmond, Virginia, USA

    Andrei I. Ivanov

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Khmelinskii, A., Knop, M. (2014). Analysis of Protein Dynamics with Tandem Fluorescent Protein Timers. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_13

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  • DOI: https://doi.org/10.1007/978-1-4939-0944-5_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0943-8

  • Online ISBN: 978-1-4939-0944-5

  • eBook Packages: Springer Protocols

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