PMID- 17016546
OWN - NLM
STAT- PubMed-not-MEDLINE
DCOM- 20070618
LR  - 20181113
IS  - 1550-7629 (Electronic)
IS  - 1550-7629 (Linking)
VI  - 4
DP  - 2006 Jul 26
TI  - Measuring ligand-dependent and ligand-independent interactions between nuclear 
      receptors and associated proteins using Bioluminescence Resonance Energy Transfer 
      (BRET).
PG  - e021
LID - e021
AB  - 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.
FAU - Koterba, Kristen L
AU  - Koterba KL
AD  - Department of Biochemistry and Cancer Biology, Medical University of Ohio, 
      Toledo, OH, USA.
FAU - Rowan, Brian G
AU  - Rowan BG
LA  - eng
PT  - Journal Article
DEP - 20060726
PL  - United States
TA  - Nucl Recept Signal
JT  - Nuclear receptor signaling
JID - 101237902
PMC - PMC1555634
EDAT- 2006/10/04 09:00
MHDA- 2006/10/04 09:01
PMCR- 2006/07/26
CRDT- 2006/10/04 09:00
PHST- 2006/01/13 00:00 [received]
PHST- 2006/05/24 00:00 [accepted]
PHST- 2006/10/04 09:00 [pubmed]
PHST- 2006/10/04 09:01 [medline]
PHST- 2006/10/04 09:00 [entrez]
PHST- 2006/07/26 00:00 [pmc-release]
AID - 10.1621/nrs.04021 [doi]
PST - epublish
SO  - Nucl Recept Signal. 2006 Jul 26;4:e021. doi: 10.1621/nrs.04021.