Native Chromatin Immunoprecipitation View Full Text


Ontology type: schema:Chapter     


Chapter Info

DATE

2004-09-01

AUTHORS

Trygve O. Tollefsbol , Alan W. Thorne , Fiona A. Myers , Tim R. Hebbes

ABSTRACT

Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic location of modified histones and other chromatin-associated factors. Here we describe the methodology we have used in our laboratory for the immunoprecipitation of chromatin isolated from cells in the absence of crosslinking. Chromatin released from nuclei by micrococcal nuclease digestion is centrifuged through sucrose gradients to allow selection of monoor dinucleosomes. This allows a protein or modification at a particular gene or locus to be mapped at higher resolution than in a crosslinked ChIP experiment. Two methods for the immunoprecipitation of chromatin are described: a large-scale fractionation by which it is possible to visualize the proteins of the immunoprecipitate by polyacrylamide gel electrophoresis, PAGE and a small-scale method that is more appropriate when the quantity of chromatin is limited. The sequence content of DNA extracted from the immunoprecipitated chromatin is analyzed by hybridization of Southern or slot blots, or by quantitative polymerase chain reaction. Enrichment of particular sequences in the immunoprecipitated fraction reveals the presence and extent of the modification at this location. More... »

PAGES

021-044

Book

TITLE

Epigenetics Protocols

ISBN

1-59259-828-5

Author Affiliations

Identifiers

URI

http://scigraph.springernature.com/pub.10.1385/1-59259-828-5:021

DOI

http://dx.doi.org/10.1385/1-59259-828-5:021

DIMENSIONS

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