Analysis of Protein Interactions In Vivo with Fluorescence Resonance Energy Transfer (FRET)

INTRODUCTIONThe protocol presented here focuses on a standard microscope-based fluorescence resonance energy transfer (FRET) assay for budding yeast, but many aspects are applicable to other systems and assays. It provides information on experimental design and data analysis procedures that can be extended to FRET experiments in any system, including mammalian cell lines and other model organisms. The steps describing strain construction can be applied to studies in yeast using fluorimetry instead of microscopy. Although the data acquisition and analysis described in this protocol can be a daunting task, careful quantitative analysis is absolutely necessary because of a high degree of cross-talk and many unknown parameters that vary from cell to cell.