Detection of ERalpha-SRC-1 interactions using bioluminescent resonance energy transfer.
Bioluminescent Resonance Energy Transfer is a naturally occurring phenomenon that can be exploited to explore protein-protein interactions in real-time in intact cells and cellular extracts. It detects energy transferred between a bioluminescent donor enzyme (Renilla luciferase) fusion protein and a fluorescent (GFP(2), a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET(2) energy transfer relies on the distance and orientation generated by the fusion proteins. This chapter describes in detail the BRET(2) assay as it is used to examine the physical interaction between the nuclear receptor ERalpha and the transcriptional coactivator SRC-1. Description of methods include selection of donor and acceptor combinations, fusion construct generation and validation, cell culture and transfection, individual fluorescence and luminescence detection, BRET(2) detection, and data analysis.
Duplessis
Tamika T
TT
Department of Structural & Cellular Biology and Tulane, University School of Medicine, New Orleans, LA, USA.
Koterba
Kristen L
KL
Rowan
Brian G
BG
D000818:Animals; D002460:Cell Line; D004735:Energy Transfer; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D006801:Humans; D049449:Luminescence; D056920:Nuclear Receptor Coactivator 1 / Q000378:metabolism*; D011485:Protein Binding
D047628:Estrogen Receptor alpha; D056920:Nuclear Receptor Coactivator 1