Chromatin immunoprecipitation to study protein-DNA interactions in budding yeast.
The accurate replication and expression of genetic information is ultimately governed by the interaction of regulatory proteins with specific sites on chromosomes. In recent years, our understanding of how these interactions occur in vivo has advanced considerably, in large part owing to the widespread application of chromatin immunoprecipitation (ChIP), a technique that allows quantification of protein-DNA interactions within the context of native chromatin. The ChIP assay involves three main steps: (1) chemical crosslinking of protein-DNA complexes in intact cells; (2) recovery of specific proteins by immunoprecipitation; and (3) detection of co-precipitating DNA sequences, usually by the polymerase chain reaction (PCR). Here, we provide a detailed description of a ChIP procedure that is commonly used to detect protein-DNA interactions in the yeast Saccharomyces cerevisiae, and discuss various methods for quantifying co-precipitating DNAs.
Ezhkova
Elena
E
Watson School of Biological Sciences, Cold Spring Harbor Laboratory, NY, USA.
Tansey
William P
WP
D002843:Chromatin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009172:Mycology / Q000379:methods; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism
D002843:Chromatin; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins