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...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166721006651 |
| _version_ | 1819095686214320128 |
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| author | Kyle Brown Samantha Kent Xiaojun Ren |
| author_facet | Kyle Brown Samantha Kent Xiaojun Ren |
| author_sort | Kyle Brown |
| collection | DOAJ |
| 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). |
| first_indexed | 2024-12-21T23:47:15Z |
| format | Article |
| id | doaj.art-c614c8de3f504d0cac585ccee3d1f128 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-21T23:47:15Z |
| publishDate | 2021-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-c614c8de3f504d0cac585ccee3d1f1282022-12-21T18:46:04ZengElsevierSTAR Protocols2666-16672021-12-0124100959Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule trackingKyle Brown0Samantha Kent1Xiaojun Ren2University of Colorado Denver Chemistry Department, 1150 12th St., Denver, CO 80204, USAUniversity of Colorado Denver Chemistry Department, 1150 12th St., Denver, CO 80204, USAUniversity of Colorado Denver Chemistry Department, 1150 12th St., Denver, CO 80204, USA; Corresponding authorSummary: 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).http://www.sciencedirect.com/science/article/pii/S2666166721006651BiophysicsSingle-molecule AssaysCell BiologyCell-based AssaysMicroscopyGene Expression |
| spellingShingle | Kyle Brown Samantha Kent Xiaojun Ren Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking STAR Protocols Biophysics Single-molecule Assays Cell Biology Cell-based Assays Microscopy Gene Expression |
| title | 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_short | 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 |
| topic | Biophysics Single-molecule Assays Cell Biology Cell-based Assays Microscopy Gene Expression |
| url | http://www.sciencedirect.com/science/article/pii/S2666166721006651 |
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