Native chromatin immunoprecipitation.
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 mono- or 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.
Institute of Biomedical and Biomolecular Sciences, School of Biological Sciences, University of Portsmouth, UK.
Alan W
AW
Thorne
Fiona A
FA
Myers
Tim R
TR
Hebbes
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D002478:Cells, Cultured; D002645:Chickens / Q000235:genetics; D002843:Chromatin / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D004247:DNA / Q000032:analysis; D004912:Erythrocytes / Q000737:chemistry / Q000502:physiology; D006657:Histones / Q000737:chemistry; D006801:Humans; D020014:K562 Cells; D016133:Polymerase Chain Reaction / Q000379:methods; D011233:Precipitin Tests / Q000379:methods*
D000906:Antibodies; D002843:Chromatin; D006657:Histones; D004247:DNA