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

ABSTRACT: 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).

SUBMITTER: Brown K

PROVIDER: S-EPMC8605088 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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

Brown Kyle K Kent Samantha S Ren Xiaojun X

STAR protocols 20211116 4

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 s ...[more]

PMID: 34825219

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