A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP

2001 ◽  
Vol 264 (5) ◽  
pp. 578-587 ◽  
Author(s):  
Y. Wang ◽  
G. Wang ◽  
D. J. O'Kane ◽  
A. A. Szalay
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Renilla Luciferase ◽  
Protein Protein Interactions

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

scholarly journals Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells

2010 ◽  
Vol 107 (31) ◽  
pp. 13582-13587 ◽  
Author(s):  
H. E. Rajapakse ◽  
N. Gahlaut ◽  
S. Mohandessi ◽  
D. Yu ◽  
J. R. Turner ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Protein Protein Interactions ◽  
Time Resolved

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

2019 ◽  
Vol 14 (4) ◽  
pp. 1084-1107 ◽  
Author(s):  
Hiroyuki Kobayashi ◽  
Louis-Philippe Picard ◽  
Anne-Marie Schönegge ◽  
Michel Bouvier
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions
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