Sequential Chromatin Immunoprecipitation Assay and Analysis

Sequential chromatin immunoprecipitation (SeqChIP) assays have been developed for the study of interactions of two or more proteins (or simultaneous histone modifications) at genomic sites. It is based on the principle that chromatin and associated proteins can be first immunoprecipitated with a first antibody and the obtained immunoprecipitate can be subjected to a second antibody. At the end of the assay the immunoprecipitated material contains only chromatin that concomitantly carries both DNA-associated proteins (or both histone modifications). The SeqChIP protocol described here combines speed (minimum of 3–4 h to perform the complete assay), sensitivity (known targets can be detected with only about 20,000 cell equivalents), and avoidance of antibody–antigen disruption after the first ChIP step. In addition, specific SeqChIP controls and potential shortcomings are discussed, the main characteristics of different SeqChIP protocols are described and several examples of protein complexes and protein–protein interactions at genomic sites that have been solved by SeqChIP in the recent years are presented.