Chromatin Immunoprecipitation:

The interaction between proteins and DNA is experimented in invitro by immuno precipitation method called ‘chromatin immunoprecipitation’. This method helps in prediction of protein specificity with genomic regions of its loci. For instance, specific protein interaction is traced in case of transcription factors involved during transcription of DNA to mRNA and then to protein. It is represented as ChIP.

Types of chromatin immuno precipitation

Based on preparation of chromatin, they are of two types

• Cross linked chromatin immuno precipitation (XChIP)
• Native chromatin immuno precipitation (NChIP)

Cross linked ChIP

Cross linked ChIP are sheared by sonication method and the DNA fragments of 3000-10000 base pairs in length. It helps in proper mapping of DNA. Reversible cross linked agents, for instance, UV light and formaldehyde to target transcription factors. Chromatin is sheared by nuclease digestion after addition of formaldehyde agent. Chromatin shears releases cell debris and are cleaned by sedimentation. However DNA and protein complex are immunologically precipitated with the help of particular antibodies according to the protein of interest. These particular antibodies get attached to magnetic beads, sepharose and or agarose. Magnetic bead, antibody, DNA sequence and protein sequence forms a complex called immune precipitated complex. These complexes are collected separately and are washed to get rid of unbound and non-specific chromatin.  After that, DNA protein cross-over takes place by inversion. Consequently, digestion of proteins occurs with the help of enzyme like proteinase K. Immuno precipitated complex is purified and DNA is analyzed by various techniques like Polymerase Chain Reaction, cloning and direct high throughput and DNA sequencing, microarray.

Native ChIP

Native ChIP helps in proper mapping of DNA protein target complex mainly on histone modifiers. Starting chromatin is Native ChIP. Histone is naturally bound over nucleosomes. In native ChIP, chromatin is sheared by digestion process called ‘micrococcal nuclease digestion’. This enzyme Micrococcal nuclease breaks DNA in to fragments of 200 – 1000 bp in length as separate nucleosomes. Cell debris is cleared as similar in ChIP type. Proteins of specific interest are precipitated from the complex. DNA associated with the complexes are purified and analyzed by sequencing techniques.