Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly define cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of epigenomics and learning more about epigenetic phenomena. Briefly, the conventional method is as follows: DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP). The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion. Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody. The associated DNA fragments are purified and their sequence is determined. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. There are mainly two types of ChIP, primarily differing in the starting chromatin preparation. The first uses reversibly cross-linked chromatin sheared by sonication called cross-linked ChIP (XChIP). Native ChIP (NChIP) uses native chromatin sheared by micrococcal nuclease digestion. Cross-linked ChIP is mainly suited for mapping the DNA target of transcription factors or other chromatin-associated proteins, and uses reversibly cross-linked chromatin as starting material. The agent for reversible cross-linking could be formaldehyde or UV light. Then the cross-linked chromatin is usually sheared by sonication, providing fragments of 300 - 1000 base pairs (bp) in length. Mild formaldehyde crosslinking followed by nuclease digestion has been used to shear the chromatin.