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The Nucleus pp 363-385 | Cite as

Fluorescence Recovery After Photobleaching (FRAP) to Study Nuclear Protein Dynamics in Living Cells

  • Martin E. van Royen
  • Pascal Farla
  • Karin A. Mattern
  • Bart Geverts
  • Jan Trapman
  • Adriaan B. Houtsmuller
Part of the Methods in Molecular Biology book series (MIMB, volume 464)

Abstract

Proteins involved in chromatin-interacting processes, like gene transcription, DNA replication, and DNA repair, bind directly or indirectly to DNA, leading to their immobilisation. However, to reach their target sites in the DNA the proteins have to somehow move through the nucleus. Fluorescence recovery after photobleaching (FRAP) has been shown to be a strong approach to study exactly these properties, i.e. mobility and (transient) immobilisation of the proteins under investigation. Here, we provide and discuss detailed protocols for some of the FRAP procedures that we have used to study protein behaviour in living cell nuclei. In addition, we provide examples of their application in the investigation of the androgen receptor (AR), a hormone-inducible transcription factor, and of two DNA-maintenance factors, the telomere binding proteins TRF1 and TRF2. We also provide protocols for qualitative FRAP analysis and a general scheme for computer modelling of the presented FRAP procedures that can be used to quantitatively analyse experimental FRAP curves.

Keywords

Fluorescence recovery after photobleaching FRAP Protein mobility Confocal microscopy Androgen receptor Fluorescent proteins 

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Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Martin E. van Royen
    • 1
  • Pascal Farla
    • 1
  • Karin A. Mattern
    • 1
  • Bart Geverts
    • 1
  • Jan Trapman
    • 1
  • Adriaan B. Houtsmuller
    • 1
  1. 1.Department of Pathology, Josephine Nefkens InstituteErasmus University Medical CentreRotterdamThe Netherlands

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