Chromatin immunoprecipitation (ChIP) is the process by which a protein of interest binds to a specific genomic DNA region. The condensed DNA and histone protein complex that is packaged together to form nucleosomes is known as chromatin. Using a ChIP assay, researchers are able to investigate the interaction certain proteins or modifications may have with DNA, and uncover the specific DNA sequence this interaction is occurring.
ChIP sequencing (ChIP-seq) combines the ChIP method with high throughput sequencing to pinpoint the exact gene sequencing to which a protein binds in living cells. Various cellular functions including gene transcription, signal transduction, chromosome segregation, and epigenetic silencing can be better understood through the assessment of protein-DNA interactions. Researchers can compare two different sample groups such as those that are healthy or diseased and determine the difference in histone methylation levels associated with a specific gene promoter region. In addition, the genomic location of histone modifications or other epigenetic mechanisms can be mapped using chromatin immunoprecipitation. Genetic targets of DNA-binding proteins and the relevant epigenetic mechanisms are important to understand in order to uncover the nuances of cellular processes.
ChIP is one of the most popular ways for investigating protein-DNA interactions of interest and gaining insight into epigenetic modifications in living cells. Certain details of a ChIP protocol can be modified in select ways, but the overarching procedure is outlined below. This protocol illustrates a standard crosslinking ChIP (X-ChIP) procedure for use in mammalian cells:
- Lyse cells with nuclear lysis buffers
- Shear chromatin
- Pre-incubate lysate with magnetic beads to reduce nonspecific immunoprecipitation
- Immunoprecipitate with an antibody
- Bind protein A/G beads to the antibody and incubate
- Magnetically precipitate beads and wash to remove weak binding
- Reverse and de-crosslink by applying heat
- Purify DNA fragments and analyze by PCR or real-time PCR
- Sequence DNA with Illumina instrument
ChIP can also be performed without cross-linking to investigate stable protein-DNA interactions, which is known as native ChIP or N-ChIP. This process, however, requires that the histones associated with DNA have atypically high stability and researchers run the risk of nucleosomes rearranging while the chromatin is being processed. X-ChIP is typically used most frequently.