Detection of Protein-Protein Interactions in vivo Using Cyan and Yellow Fluorescent Proteins
This article describes a technique for detecting protein?protein interactions in vivo using cyan and yellow fluorescent proteins. The phenomenon of fluorescence resonance energy transfer (FRET) describes the transfer of energy from one fluorophore to another through dipole-dipole interaction. The use of GFP variants such as CFP (cyan) and YFP (yellow) in FRET analysis has the additional advantage of allowing detection of intracellular associations in living cells. While most of the applications of FRET have employed fluorescence microscopic imaging methods, it has recently become feasible to perform FRET analysis using flow cytometry. One needs to perform electronic compensation to remove CFP emission from the FRET channel (P5-P6). One then should perform interlaser compensation between CFP and YFP using Omnicomp circuitry to remove YFP excitation from the 413-nm laser. Analyze collected data using Flowjo software. Control samples transfected with non-interacting FRET pairs should be used as negative controls to determine the baseline FRET signal. © 2006 Copyright © 2006 Elsevier Inc. All rights reserved.
