Authors
Ignacio Demarco, Cynthia Booker, and Richard Day
Summary
A variety of live-cell imaging methods are available that provide important insights into the dynamic behavior of proteins in intact cells. Further, FRET-based microscopy techniques provide a way to gain the angstrom-scale resolution that is necessary to detect protein-protein interactions. This protocol details a live-cell imaging method that combines dynamic measurements made possible by the photoactivated green fluorescent protein (PA-GFP) with measurements on the scale of angstroms using Förster resonance energy transfer (FRET) microscopy1. This new method, called photo-quenching FRET (PQ-FRET) exploits the quenching of the fluorescence from a donor fluorophore when there is energy transfer to nearby acceptor fluorophores. The PQ-FRET method uses PA-GFP as a photoactivatable FRET acceptor, and monitors the attendant quenching of cyan FP (CFP)-labeled donor proteins, allowing the dynamics of interactions between proteins fused to the fluorophores to be quantified. As an example, we use PQ-FRET here to demonstrate the dynamic association of heterochromatin protein-1 alpha (HP1α) with the transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) within the living cell nucleus. The PQ-FRET assay provides direct measurements of protein mobility, exchange and interactions within macromolecular complexes in living cells without the need for corrections based on reference images acquired from separate control cells.Further details
The protocol was published on Protocol Exchange in 2006. To see the entire protocol, click on the source link.Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 736
- Comments 0