Immunoprecipitation of chromatin

Abstract

This chapter discusses the immunoprecipitation of chromatin. The chapter discusses three standard protocols, all of which are used to analyze the role of specific proteins within defined regions of the eukaryotic genome. It analyzes both the DNA and the protein after immunoprecipitation. This requires a careful examination of the protocol chosen as not all are amenable to protein analysis. The conformation of protein antigens is likely to vary depending on whether they are in their native state in solution, formaldehyde-fixed, bound to nitrocellulose filters, or fixed to plastic microtiter plates. Nonspecific binding of the antibody to chromatin should also be carefully controlled. Antibodies may be added in excess if complete depletion of the target protein is required. Affinity purification of antisera also minimizes background and, if possible, should be routinely carried out. Some studies requiring the precise positioning of DNA-binding proteins are hampered by the exchange, sliding, or artificial movements of proteins during chromatin isolation. Treatment with formaldehyde dimethyl sulfate/borohydride or UV irradiation efficiently cross-links proteins to DNA. Methods for formaldehyde cross-linking of chromatin in nuclei is described in detail.