Nature Methods 3, 519 - 524 (2006)
Published online: 21 June 2006; | doi:10.1038/nmeth889
Monitoring dynamic protein interactions with photoquenching FRETIgnacio A Demarco1, Ammasi Periasamy2, Cynthia F Booker1
& Richard N Day11
Departments of Medicine and Cell Biology, P.O. Box 800578, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908, USA. 2
W.M. Keck Center for Cellular Imaging, University of Virginia, Gilmer Hall, Charlottesville, Virginia, 22904, USA.
Correspondence should be addressed to Richard N Day rnd2v@virginia.edu 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 (HP1 ) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBP) 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.
MORE ARTICLES LIKE THIS These links to content published by NPG are automatically generated.
|
1.2 NA water-immersion objective lens (Olympus)
