Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy

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Item Type:	Review
Title:	Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy
Creators Name:	Phair, R.D., Gorski, S.A. and Misteli, T.
Abstract:	Chromatin-binding proteins play a crucial part in every aspect of chromatin structure and gene expression. An experimental approach to studying the binding of protein to chromatin in living cells is the use of photobleaching methods. In these experiments a fluorescently tagged protein of interest is introduced into cells and its apparent mobility is measured as an indicator of its dynamic properties. Because nuclear proteins move passively by rapid diffusion through the nuclear space, binding of a protein dramatically affects its overall mobility and therefore the measured mobility contains information about the in vivo binding properties of a protein. Qualitative analysis of photobleaching data gives an impression of whether a protein binds stably or transiently to chromatin in vivo. In addition to the standard qualitative analysis of photobleaching experiments, kinetic modeling methods can be applied for data analysis to permit extraction of quantitative information about simple biophysical properties of chromatin proteins. This method can be used to determine the residence time of a protein on chromatin, the size of the bound and free pools, and the number of kinetically distinct fractions of a protein.
Keywords:	Binding Sites, Biochemistry, Biological Models, Cell Nucleus, Chromatin, Computer-Assisted Image Processing, Cytoplasm, Fluorescence Microscopy, Fluorescence Recovery After Photobleaching, Green Fluorescent Proteins, Kinetics, Luminescent Proteins, Protein Binding, Time Factors
Source:	Methods in Enzymology
Title of Book:	Chromatin and Chromatin Remodeling Enzymes, Part A
ISSN:	0076-6879
Publisher:	Academic Press
Volume:	375
Page Range:	393-414
Date:	2003
Official Publication:	https://doi.org/10.1016/s0076-6879(03)75025-3
PubMed:	View item in PubMed

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