title: Fluorescence Resonance Energy Transfer Microscopy for Measuring Chromatin Complex Structure and Dynamics.
abstract: The Polycomb group (PcG) proteins form regulatory complexes that modify the chromatin structure and silence their target genes. Recent works have found that the composition of Polycomb complexes is highly dynamic. Defining the different protein components of each complex is fundamental for better understanding their biological functions. Fluorescent resonance energy transfer (FRET) is a powerful tool to measure protein-protein interactions, in nanometer order and in their native cellular environment. Here we describe the preparation and execution of a typical FRET experiment using CFP-tagged protein as donor and YFP-tagged protein as acceptor. We further show that FRET can be used in a competition assay to measure binding affinities of different components of the same chromatin complex.
authors.affiliation: Fondazione Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi", Via Francesco Sforza 35, 20122, Milan, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Cherubini
authors.affiliation: Fondazione Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi", Via Francesco Sforza 35, 20122, Milan, Italy. zippo@ingm.org.
authors.forename: Alessio
authors.initials: A
authors.lastname: Zippo
mesh_terms: D002843:Chromatin / Q000235:genetics / Q000302:isolation & purification*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D038761:Photobleaching; D063146:Polycomb-Group Proteins / Q000235:genetics / Q000302:isolation & purification*; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D002843:Chromatin; D063146:Polycomb-Group Proteins; D049452:Green Fluorescent Proteins
keywords: Acceptor photobleaching; CFP; Confocal microscopy; Fluorescence resonance energy transfer (FRET); PcG; YFP