Monitoring dynamic protein interactions with photoquenching FRET

Ignacio A. Demarco, Ammasi Periasamy, Cynthia F. Booker, Richard N. Day

Research output: Contribution to journalArticlepeer-review

75 Scopus citations

Abstract

The mammalian cell nucleus is a dynamic and highly organized structure. Most proteins are mobile within the nuclear compartment, and this mobility reflects transient interactions with chromatin, as well as network interactions with a variety of protein partners. To study these dynamic processes in living cells, we developed an imaging method that combines the photoactivated green fluorescent protein (PA-GFP) and fluorescence resonance energy transfer (FRET) microscopy. We used this new method, photoquenching FRET (PQ-FRET), to define the dynamic interactions of the heterochromatin protein-1 alpha (HP1α) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) in regions of centromeric heterochromatin in mouse pituitary cells. The advantage of the PQ-FRET assay is that it provides simultaneous measurement of a protein's mobility, its exchange within macromolecular complexes and its interactions with other proteins in the living cell without the need for corrections based on reference images acquired from control cells.

Original languageEnglish (US)
Pages (from-to)519-524
Number of pages6
JournalNature Methods
Volume3
Issue number7
DOIs
StatePublished - Jul 2006

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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