Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking

Summary: This protocol provides instructions to track the global dynamics of single epigenetic regulatory factors in live cells. We describe an approach to generate cell lines that stably express HaloTag-fused proteins. We then use live-cell single-molecule tracking to obtain kinetic populations and...

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Autores principales:	Kyle Brown, Samantha Kent, Xiaojun Ren
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Biophysics Single-molecule Assays Cell Biology Cell-based Assays Microscopy Gene Expression Science (General) Q1-390
Acceso en línea:	https://doaj.org/article/c614c8de3f504d0cac585ccee3d1f128
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spelling	oai:doaj.org-article:c614c8de3f504d0cac585ccee3d1f1282021-11-18T04:51:43ZQuantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking2666-166710.1016/j.xpro.2021.100959https://doaj.org/article/c614c8de3f504d0cac585ccee3d1f1282021-12-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2666166721006651https://doaj.org/toc/2666-1667Summary: This protocol provides instructions to track the global dynamics of single epigenetic regulatory factors in live cells. We describe an approach to generate cell lines that stably express HaloTag-fused proteins. We then use live-cell single-molecule tracking to obtain kinetic populations and residence times. The kinetic parameters obtained can be used to determine important aspects of transcriptional regulation such as target-search time, 3D free diffusion time, and number of non-specific sites sampled before reaching a specific site and compare behaviors across different nuclear environments.For complete details on the use and execution of this protocol, please refer to Kent et al. (2020).Kyle BrownSamantha KentXiaojun RenElsevierarticleBiophysicsSingle-molecule AssaysCell BiologyCell-based AssaysMicroscopyGene ExpressionScience (General)Q1-390ENSTAR Protocols, Vol 2, Iss 4, Pp 100959- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biophysics Single-molecule Assays Cell Biology Cell-based Assays Microscopy Gene Expression Science (General) Q1-390
spellingShingle	Biophysics Single-molecule Assays Cell Biology Cell-based Assays Microscopy Gene Expression Science (General) Q1-390 Kyle Brown Samantha Kent Xiaojun Ren Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking
description	Summary: This protocol provides instructions to track the global dynamics of single epigenetic regulatory factors in live cells. We describe an approach to generate cell lines that stably express HaloTag-fused proteins. We then use live-cell single-molecule tracking to obtain kinetic populations and residence times. The kinetic parameters obtained can be used to determine important aspects of transcriptional regulation such as target-search time, 3D free diffusion time, and number of non-specific sites sampled before reaching a specific site and compare behaviors across different nuclear environments.For complete details on the use and execution of this protocol, please refer to Kent et al. (2020).
format	article
author	Kyle Brown Samantha Kent Xiaojun Ren
author_facet	Kyle Brown Samantha Kent Xiaojun Ren
author_sort	Kyle Brown
title	Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking
title_short	Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking
title_full	Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking
title_fullStr	Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking
title_full_unstemmed	Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking
title_sort	quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/c614c8de3f504d0cac585ccee3d1f128
work_keys_str_mv	AT kylebrown quantifyingtheglobalbindingandtargetsearchdynamicsofepigeneticregulatoryfactorsusinglivecellsinglemoleculetracking AT samanthakent quantifyingtheglobalbindingandtargetsearchdynamicsofepigeneticregulatoryfactorsusinglivecellsinglemoleculetracking AT xiaojunren quantifyingtheglobalbindingandtargetsearchdynamicsofepigeneticregulatoryfactorsusinglivecellsinglemoleculetracking
_version_	1718425002761519104

Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking

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