Detection of ER-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 (GFP² , a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET² energy transfer relies on the distance and orientation generated by the fusion proteins. This chapter describes in detail the BRET² assay as it is used to examine the physical interaction between the nuclear receptor ERα 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, BRET² detection, and data analysis.