12 Measuring the Proximity of Proteins in Living Cells by Fluorescence Resonance Energy Transfer between CFP and YFP

Abstract

Publisher Summary This chapter describes the complete process of a fluorescence resonance energy transfer (FRET)-based analysis of the organization of a protein complex. It also compares the properties of the two versions of yellow fluorescent protein (YFP) for their value as FRET donors in yeast. Protocols for tagging proteins are also presented in the chapter. FRET is measured by digital epifluorescence microscopy. The chapter demonstrates the use of FRET R to measure sensitized emission. FRET R is both intuitive, easy to implement, and simplifies comparisons across projects and labs. Fluorescence microscopy can determine whether two tagged proteins localize to the same compartment, but because of the limit of the resolution of light microscopy, it cannot demonstrate interactions between a protein pair. FRET overcomes this limitation because typically FRET can only occur if two fluorophores are at least within 7nm. FRET is the transfer of excitation energy from one fluorophore, referred to as “the donor,” to a second fluorophore, the “acceptor.” It can occur when the wave properties of the electrons in the excited state of the donor overlap the wave properties of the ground state electrons of the acceptor.