Characterization of protein-DNA association in vivo by chromatin immunoprecipitation.
Chromatin immunoprecipitation (ChIP) is one of the most powerful methods to identify and characterize the association of proteins with specific genomic regions in the context of intact cells. In this method, cells are first treated with formaldehyde to crosslink protein-protein and protein-DNA complexes in situ. Next, the crosslinked chromatin is sheared by sonication to generate small chromatin fragments, and the fragments associated with the protein of interest are immunoprecipitated using antibodies to the protein. Finally, protein-DNA crosslinks are reversed and the DNA is examined for the presence of particular sequences by quantitative polymerase chain reaction (PCR). Enrichment of specific sequences in the precipitate indicates that the sequences are associated with the protein of interest in vivo. The ChIP method described here is intended for studying protein-DNA association in the budding yeast Saccharomyces cerevisiae, but it can be easily implemented in other cell types, including fly, mammalian, and plant cells.
Kuras
Laurent
L
Centre de Genetique Moleculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
D000818:Animals; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D005557:Formaldehyde / Q000737:chemistry; D008863:Microspheres; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests / Q000379:methods*; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000378:metabolism; D013205:Staphylococcal Protein A
D002843:Chromatin; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D013205:Staphylococcal Protein A; D005557:Formaldehyde; D004247:DNA