Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking
| Autor: | Kyle Brown, Samantha Kent, Xiaojun Ren |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Science (General)
Computer science Cell Biophysics Gene Expression Computational biology Tracking (particle physics) General Biochemistry Genetics and Molecular Biology Epigenesis Genetic Q1-390 Protocol medicine Transcriptional regulation Humans Single-molecule Assays Epigenetics Free diffusion Microscopy General Immunology and Microbiology General Neuroscience Dynamics (mechanics) Cell Biology Cell based assays Single Molecule Imaging Kinetics HEK293 Cells medicine.anatomical_structure Gene Expression Regulation Molecular/Chemical Probes Cell-based Assays Single-Cell Analysis Protein Binding |
| Zdroj: | STAR Protocols, Vol 2, Iss 4, Pp 100959-(2021) STAR Protocols |
| ISSN: | 2666-1667 |
| Popis: | 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). Graphical abstract Highlights • Generate cell lines stably expressing HaloTag fusion genes • Quantify global binding and target-search kinetics of epigenetic factors • Map binding dynamics of epigenetic factors within transcriptional condensates 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. |
| Databáze: | OpenAIRE |
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