Chromatin Immunoprecipitation (ChIP)

Abstract

INTRODUCTIONChromatin immunoprecipitation (ChIP) is an invaluable method for studying interactions between specific proteins or modified forms of proteins and a genomic DNA region. ChIP can be used to determine whether a transcription factor interacts with a candidate target gene and is used with equal frequency to monitor the presence of histones with post-translational modifications at specific genomic locations. In early ChIP studies, UV light from a transilluminator was used to cross-link proteins to DNA irreversibly. The cross-linked chromatin was then either sonicated or cleaved with restriction enzymes to generate smaller DNA fragments, followed by immunoprecipitation with the desired antibodies. The precipitated protein-DNA adducts were then purified, treated with a protease, and analyzed by dot blot or Southern blot using a radiolabeled probe derived from the cloned DNA fragment of interest. The use of formaldehyde as a reversible protein-DNA and protein-protein cross-linking agent for ChIP and the use of polymerase chain reaction (PCR) to detect precipitated DNA fragments were later added as components of the modern ChIP procedure. The protocol below represents a standard ChIP procedure for use in mammalian cells. Cross-linking is performed by adding formaldehyde to growing cells, and chromatin is prepared, sheared by sonication, and precleared to reduce nonspecific immunoprecipitation. Immunoprecipitation is performed with a specific antibody. After elution of the protein-DNA complexes from protein A- or protein G-agarose resin, the samples are heated to reverse the covalent cross-links. The DNA fragments are purified and analyzed by PCR or real-time PCR.