title: Monitoring dynamic binding of chromatin proteins in vivo by fluorescence correlation spectroscopy and temporal image correlation spectroscopy.
abstract: Live-cell microscopy has demonstrated that many nuclear proteins bind transiently to target sites in chromatin. These binding interactions can be detected and quantified by two related live-cell imaging techniques, Fluorescence Correlation Spectroscopy (FCS) and Temporal Image Correlation Spectroscopy (TICS). With proper quantitative modeling, it is possible to obtain estimates from FCS and TICS data of the association and dissociation rates of nuclear protein binding to chromatin. These binding rates permit calculating the fractions of free and bound protein in the nucleus, plus the time required to diffuse from one binding site to the next and the dwell time on a chromatin target. In this protocol, we summarize the underlying principles of FCS and TICS, and then describe how these data should be collected and analyzed to extract estimates of in vivo binding.
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Davide
authors.initials: D
authors.lastname: Mazza
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Stasevich
authors.forename: Tatiana S
authors.initials: TS
authors.lastname: Karpova
authors.forename: James G
authors.initials: JG
authors.lastname: McNally
mesh_terms: D002138:Calibration; D002470:Cell Survival; D002843:Chromatin / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013223:Statistics as Topic; D013997:Time Factors
chemical_list: D002843:Chromatin; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins