Bioluminescence resonance energy transfer-based imaging of protein-protein interactions in living cells

Autor: Hiroyuki, Kobayashi, Louis-Philippe, Picard, Anne-Marie, Schönegge, Michel, Bouvier
Rok vydání: 2018
Předmět:
Zdroj: Nature protocols. 14(4)
ISSN: 1750-2799
Popis: Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein-protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP). Because the BRET donor does not require an external light source, it does not lead to phototoxicity or autofluorescence. It therefore represents an interesting alternative to fluorescence-based imaging such as FRET. However, the low signal output of BRET energy donors has limited the spatiotemporal resolution of BRET imaging. Here, we describe how recent improvements in detection devices and BRET probes can be used to markedly improve the resolution of BRET imaging, thus widening the field of BRET imaging applications. The protocol described herein involves three main stages. First, cell preparation and transfection require 3 d, including cell culture time. Second, image acquisition takes 10-120 min per sample, after an initial 60 min for microscope setup. Finally, image analysis typically takes 1-2 h. The choices of energy donor, acceptor, luminescent substrates, cameras and microscope setup, as well as acquisition modes to be used for different applications, are also discussed.
Databáze: OpenAIRE
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