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Fluorescence Recovery After Photobleaching

Part of the Methods in Molecular Biology book series (MIMB,volume 769)

Abstract

This chapter describes the use of microscope-based fluorescence recovery after photobleaching (FRAP). To quantify the dynamics of proteins within a subcellular compartment, we first outline the general aspects of FRAP experiments and then provide a detailed protocol of how to measure and analyse the most important parameters of FRAP experiments such as mobile fraction and half-time of recovery.

Key words

  • Fluorescence recovery after photobleaching
  • Fluorescence microscopy
  • Green fluorescent protein
  • Half-time of recovery
  • Mobile fraction
  • Diffusion
  • Binding reaction kinetics
  • Focal adhesions
  • Vinculin

Alex Carisey, Matthew Stroud, and Ricky Tsang contributed equally to the manuscript.

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Acknowledgments

We would like to thank Dr. Janet Askari for critical reading of the manuscript. CB acknowledges BBSRC (BB/G004552/1). The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund.

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Correspondence to Christoph Ballestrem .

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Carisey, A., Stroud, M., Tsang, R., Ballestrem, C. (2011). Fluorescence Recovery After Photobleaching. In: Wells, C., Parsons, M. (eds) Cell Migration. Methods in Molecular Biology, vol 769. Humana Press. https://doi.org/10.1007/978-1-61779-207-6_26

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  • DOI: https://doi.org/10.1007/978-1-61779-207-6_26

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-206-9

  • Online ISBN: 978-1-61779-207-6

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