Chromatin Immunoprecipitation
The chromatin immunoprecipitation ( ChIP ) is an experimental method for the determination of protein-DNA interactions . One goal is to find out whether certain proteins are associated with specific gene regions. For example, the binding of a transcription factor to promoters , enhancers , repressors or silencers can be examined. Another important field of research is the investigation of the distribution of the various histone modifications , a basis for epigenetic gene regulation .
properties
The aim of the method is to determine whether certain proteins (mostly transcription factors ) bind certain parts of the chromatin of living cells or tissues . Since the method works with regard to crosslinking in vivo , it differs from other methods that pursue the same goal, e.g. B. the DNase Footprinting Assay . After lysis of the cells, the lysate is on so-called. Beads loaded on which the precipitation takes place. This separation takes place accordingly in vitro .
The basic principle of chromatin immunoprecipitation is based on holding the protein - DNA bonds that exist at a given point in time by fixing them with formaldehyde . The cells are then destroyed and the chromatin is shattered into pieces several hundred base pairs in length using ultrasound . Those pieces of DNA that have bound the desired protein are immunoprecipitated (isolated) with an antibody specific for the protein . The isolated DNA-protein complexes are dissolved. The identity of the respective DNA pieces can be clarified in various ways: If there is a hypothesis about the probable binding site in the genome , a PCR can be carried out using primers that are specific for the suspected DNA region. If, on the other hand, one is interested in tracing all binding sites of the protein in the entire genome, a DNA microarray can be used. In this case one also speaks of a ChIP-on-Chip experiment. Alternatively, a combination with methods known as sequencing of the second generation ( engl. Second generation sequencing ) are referred to possible ( ChIP-Seq ).
The main problem with chromatin immunoprecipitation is to find highly specific antibodies for the proteins being examined. One way is to fuse the desired protein with an epitope that is recognized by available antibodies. However, this requires a prior intervention, such as a transient transfection. Another possibility is the fusion with an amino acid chain , which are recognized by certain enzymes , which enrich certain side chains of the protein with biotin or another protein tag . The isolation of the DNA-protein complexes is made possible by the fact that the biotin in turn binds the protein streptavidin extremely specifically with extremely high affinity .
literature
- V. Orlando, Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. , Trends Biochem Sci . 2000, 25 (3) pp. 99-104.
Individual evidence
- ↑ V. Orlando, R. Paro: Mapping Polycomb-repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin. In: Cell (1993), Volume 75, Issue 6, pp. 1187-1198. PMID 7903220 .
- ↑ MJ Buck, JD Lieb: ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. In: Genomics (2004), Volume 83, Issue 3, pp. 349-360. PMID 14986705 .
- ↑ H.Kimura: “e.” In “J.Hum.Genet.” (2013), Volume 58, Issue 7, pp. 439–445. PMID 23739122
