Quantification of Protein–DNA Interactions by In Vivo Chromatin Immunoprecipitation in Yeast

Abstract

Chromatin immunoprecipitation (ChIP) is the most widely used method to measure the interaction of proteins with their target DNA sequences in the living cell. The use of ChIP can address many of the fundamental processes underlying transcription, such as the positioning and modification of nucleosomes, the binding of specific transcription factors to regulatory sequences, the secondary recruitment of chromatin-modifying complexes, and other signalling molecules to chromosomal DNA, and the occupancy of RNA polymerase complexes. ChIP is especially useful to define the dynamic nature of these processes. The basis for ChIP in most applications is the determination of the immunoprecipitation (IP) efficiency of individual genomic regions by comparing the amounts of DNA in the IP sample and the input sample before immunoprecipitation. DNA quantification relies on sensitive methods, such as quantitative PCR (qPCR), in real time. Here, we describe the methodology to perform ChIP in the yeast Saccharomyces cerevisiae in combination with qPCR in real time to determine protein–DNA association in vivo.