Individual and sequential chromatin immunoprecipitation protocols

Abstract

DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene expression making it relevant to determine the profiles of cohabitation of several proteins on the chromatin fiber. Chromatin immunoprecipitation (ChIP) has been broadly used to determine the profile of several histone posttranslational modifications as well as transcription factor occupancy in vivo. However, individual ChIP does not resolve whether the epitope under study is present at the same time on a given genomic location. Here we describe the ChIP-re-ChIP assay that represents a direct strategy to determine the in vivo co-localization of proteins or histone posttranslational modifications in a chromatinized template on the basis of double and independent rounds of immunoprecipitation with high-quality ChIP-grade antibodies.