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Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis
Published on: March 23, 2021
A fast carrier chromatin immunoprecipitation method applicable to microdissected tissue samples.
Haiping Hao1, Hester Liu, Gregory Gonye
1Daniel Baugh Institute for Functional Genomics and Computational Biology, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, United States.
Journal of Neuroscience Methods
|May 27, 2008
Summary
A new Fast Chromatin Immunoprecipitation (Fast CChIP) method enables studying transcription factor-DNA interactions in small brain tissue samples. This technique confirms cyclic-AMP response element binding (CREB) protein
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Studying transcriptional regulation in the central nervous system (CNS) is challenging due to cellular diversity and plasticity.
- Traditional methods like Chromatin Immunoprecipitation (ChIP) require larger tissue samples, limiting their application in specific neuronal populations.
- Microdissection of specific neuronal groups can reduce complexity but often excludes powerful molecular techniques.
Purpose of the Study:
- To develop a novel method for analyzing in vivo transcription factor-DNA interactions in limited CNS tissue samples.
- To adapt Chromatin Immunoprecipitation (ChIP) for use with microdissected neuronal populations.
- To investigate the role of CREB in hypertension-induced gene expression in specific brain regions.
Main Methods:
- Development of a Fast Carrier ChIP (Fast CChIP) protocol.
- Utilizing minimal brain tissue samples (as little as 0.2 mm³).
- Employing antibody against phosphorylated cyclic-AMP response element binding (CREB) protein and analyzing binding at the c-fos gene promoter.
Main Results:
- Successfully confirmed phospho-CREB (pCREB) binding at the c-fos gene promoter using Fast CChIP.
- Demonstrated hypertension-induced pCREB binding at the c-fos promoter in the rat nucleus tractus solitarius (NTS).
- Confirmed the role of CREB in mediating hypertension-induced c-fos expression.
Conclusions:
- Fast CChIP is a viable method for studying in vivo transcription factor-DNA interactions in small CNS tissue samples.
- The method allows for the investigation of specific neuronal populations and their regulatory mechanisms.
- Fast CChIP has broad applicability for research on individual brain nuclei and clinical biopsy samples.
Related Concept Videos
02:36Chromatin Immunoprecipitation- ChIP
Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or histone proteins. It also helps determine the type of histone modifications such as acetylation, phosphorylation, or methylation.
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
01:20Immunoprecipitation
Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...