Chromatin immunoprecipitation

The chromatin immunoprecipitation (ChIP ) is a biochemical method for the determination of DNA - protein interactions. DNA -protein interactions occur in binding sequences for transcription factors, promoters, enhancers, repressors, silencers, insulators, as well as binding sequences for the DNA replication.

Properties

The aim of the method is to determine whether certain proteins (mainly transcription factors ) bind certain parts of the chromatin of living cells or tissues. Since the process operates in vivo, it is different from other methods which pursue the same goal, such as DNase footprinting assay.

The basic principle of chromatin immunoprecipitation is based on retaining the existing at a time when protein-DNA binding by fixation with formaldehyde. Then the cells are disrupted and the chromatin smashed into pieces by means of ultrasound of a few hundred base pairs in length. Those pieces of DNA that have bound the desired protein are immunopräzipiert with a specific antibody for the protein (isolated). The isolated DNA protein complexes are resolved. The identity of the DNA fragments can be resolved in several ways: If you have a hypothesis about the likely binding site in the genome, it can be a PCR using primers that are specific for the putative DNA region perform. It is, however, the tracking of all of the protein binding sites in the whole genome is interested, a DNA microarray can be used. In this case, one also speaks of a ChIP -on -chip experiment.

The main problem of chromatin immunoprecipitation is to find highly specific antibodies for the proteins studied. A possibility is to fuse the desired protein having an epitope recognized by available antibodies. Another possibility is the fusion with an amino acid sequence that are recognized by specific enzymes, which accumulate specific side chains of the protein with biotin or other protein tag. The isolation of the DNA-protein complex is made possible by that the highly specific biotin in turn with an extremely high affinity protein streptavidin.