Abstract
Fundamental parameters of biological control require sequence-specific protein-deoxyribonucleic acid (DNA) interactions. The packaging of DNA into higher order chromatin controls accessibility to DNA binding proteins that influence regulatory events that include transcription, replication, recombination, and DNA repair. Chromatin is remodeled in a physiologically responsive manner by covalently modifying histones to neutralize a positive charge of these basic chromosomal proteins that form nucleosomes, the primary unit of nucleoprotein structure. Acetylation and methylation of residues on the unstructured tails of histone proteins are the most prevalent chromatin modification related to transcriptional regulation. To understand chromatin structure of a gene, it is important to characterize the interactions of structural and regulatory proteins in the vicinity of covalently modified nucleosomes. Formaldehyde crosslinking rapidly fixes protein-protein and protein-DNA complexes in vivo providing the basis for an approach to analyze native structure of gene promoters. The protocol below is a simple yet sensitive method to determine whether a known protein is associated with a DNA sequence in mammalian cells. It must be noted that this protocol is not limited to sequence-specific DNA binding proteins. The success of this procedure relies on the use of an antibody that specifically and tightly binds its target protein in the buffer, and wash conditions used for the chromatin immunoprecipitation (ChIP) assay. Briefly, the protein-protein and protein-DNA complexes in vivo are reversibly crosslinked by formaldehyde.
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© 2004 Humana Press Inc.
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Javed, A. et al. (2004). Chromatin Immunoprecipitation. In: Giordano, A., Romano, G. (eds) Cell Cycle Control and Dysregulation Protocols. Methods in Molecular Biology™, vol 285. Humana Press. https://doi.org/10.1385/1-59259-822-6:041
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DOI: https://doi.org/10.1385/1-59259-822-6:041
Publisher Name: Humana Press
Print ISBN: 978-0-89603-949-0
Online ISBN: 978-1-59259-822-9
eBook Packages: Springer Protocols