Journal
NATURE METHODS
Volume 3, Issue 7, Pages 519-524Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH889
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Funding
- NIDDK NIH HHS [R01 DK043701-14A1, R01 DK043701, DK47301] Funding Source: Medline
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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 alpha) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) 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.
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