An Optimized Chromatin Immunoprecipitation Protocol for Quantification of Protein-DNA Interactions
| Autor: | Mariel O. Brok, Patrick Kemmeren, Frank C.P. Holstege, Wim J. de Jonge |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Chromatin Immunoprecipitation
Saccharomyces cerevisiae Proteins Immunoprecipitation Saccharomyces cerevisiae Computational biology Cell fate determination Real-Time Polymerase Chain Reaction Genome General Biochemistry Genetics and Molecular Biology Article 03 medical and health sciences 0302 clinical medicine Protein Interaction Maps lcsh:Science (General) DNA Fungal Transcription factor 030304 developmental biology 0303 health sciences General Immunology and Microbiology biology Chemistry General Neuroscience biology.organism_classification Yeast Chromatin immunoprecipitation 030217 neurology & neurosurgery Function (biology) lcsh:Q1-390 |
| Zdroj: | Star Protocols STAR Protocols, Vol 1, Iss 1, Pp 100020-(2020) STAR Protocols |
| ISSN: | 2666-1667 |
| Popis: | Summary Transcription factors are important regulators of cell fate and function. Knowledge about where transcription factors are bound in the genome is crucial for understanding their function. A common method to study protein-DNA interactions is chromatin immunoprecipitation (ChIP). Here, we present a revised ChIP protocol to determine protein-DNA interactions for the yeast Saccharomyces cerevisiae. We optimized several aspects of the procedure, including cross-linking and quenching, cell lysis, and immunoprecipitation steps. This protocol facilitates sensitive and reproducible quantitation of protein-DNA interactions. For complete details on the use and execution of this protocol, please refer to (de Jonge et al., 2019). Graphical Abstract Highlights • Chromatin immunoprecipitation protocol to quantify protein-DNA interactions • Optimized for sensitivity and robustness • Optimized for quantitative comparisons between experiments, e.g., in time series • Highlights common ChIP pitfalls, variable steps, and how to increase reproducibility Transcription factors are important regulators of cell fate and function. Knowledge about where transcription factors are bound in the genome is crucial for understanding their function. A common method to study protein-DNA interactions is chromatin immunoprecipitation (ChIP). Here, we present a revised ChIP protocol to determine protein-DNA interactions for the yeast Saccharomyces cerevisiae. We optimized several aspects of the procedure, including cross-linking and quenching, cell lysis, and immunoprecipitation steps. This protocol facilitates sensitive and reproducible quantitation of protein-DNA interactions. |
| Databáze: | OpenAIRE |
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