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FRAP and FRET Methods to Study Nuclear Receptors in Living Cells

  • Protocol
The Nuclear Receptor Superfamily

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 505))

Asbract

Quantitative imaging techniques of fluorescently-tagged proteins have been instrumental in the study of the behavior of nuclear receptors (NRs) and coregulators in living cells. Ligand-activated NRs exert their function in transcription regulation by binding to specific response elements in promotor and enhancer sequences of genes. Fluorescence recovery after photobleaching (FRAP) has proven to be a powerful tool to study the mobility of fluorescently-labeled molecules in living cells. Since binding to DNA leads to the immobilization of DNA-interacting proteins like NRs, FRAP is especially useful for determining DNA-binding kinetics of these proteins. The coordinated interaction of NRs with promoters/enhancers and subsequent transcription activation is not only regulated by ligand but also by interactions with sets of cofactors and, at least in the case of the androgen receptor (AR), by dimerization and interdomain interactions. In living cells, these interactions can be studied by fluorescence resonance energy transfer (FRET).

Here we provide and discuss detailed protocols for FRAP and FRET procedures to study the behavior of nuclear receptors in living cells. On the basis of our studies of the AR, we provide protocols for two different FRAP methods (strip-FRAP and FLIP-FRAP) to quantitatively investigate DNA-interactions and for two different FRET approaches, ratio imaging, and acceptor photobleaching FRET to study AR domain interactions and interactions with cofactor motifs. Finally, we provide a protocol of a technique where FRAP and acceptor photobleaching FRET are combined to study the dynamics of interacting ARs.

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Author information

Authors and Affiliations

  1. Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands

    Martin E. van Royen, Christoffel Dinant, Pascal Farla, Jan Trapman & Adriaan B. Houtsmuller

Authors
  1. Martin E. van Royen
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  2. Christoffel Dinant
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  3. Pascal Farla
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  4. Jan Trapman
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  5. Adriaan B. Houtsmuller
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Editors and Affiliations

  1. School of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, UK

    Iain J. McEwan

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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van Royen, M.E., Dinant, C., Farla, P., Trapman, J., Houtsmuller, A.B. (2009). FRAP and FRET Methods to Study Nuclear Receptors in Living Cells. In: McEwan, I.J. (eds) The Nuclear Receptor Superfamily. Methods in Molecular Biology™, vol 505. Humana Press. https://doi.org/10.1007/978-1-60327-575-0_5

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  • DOI: https://doi.org/10.1007/978-1-60327-575-0_5

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-574-3

  • Online ISBN: 978-1-60327-575-0

  • eBook Packages: Springer Protocols

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