Sequential Chromatin Immunoprecipitation Protocol: ChIP-reChIP

Abstract

Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene transcription. Therefore, it has become relevant to determine the cohabitation of several proteins in a particular developmental stage and cell type. Furthermore, multiple post-translational histone modifications can be analyzed on the same genomic location with the aim of deciphering the combinatorial pattern of histone modifications associated to specific transcriptional stages during cell commitment. Here we describe the ChIP-reChIP assay that represents a direct strategy to determine the in vivo colocalization of proteins interacting or in close contact in a chromatinized template on the basis of double and independent rounds of immunoprecipitations with high-quality ChIP grade antibodies.