Chromatin Immunoprecipitation (ChIP) is a method of determining
the interaction between a protein of interest and a specific
sequence of DNA.
It is a powerful assay technique that can be used to probe
DNA-protein interaction within a cell's neutral chromatin
environment.
Also, this technique can provide information regarding the
repertoire of sites on DNA molecules associated with a particular
factor of transcription or histone proteins.
Information regarding precise genomic locations of various
histone modifications, such as acetylation or methylation, can also
be obtained through the Chip technique.
Spatial and temporal relationship of many of protein-DNA
interactions, can be studied through this method.
Working of Chromatin Immunoprecipitation (ChIP)
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Examination of the availability of protein-DNA interaction at
steady state, or quantification of changes in interaction at
specific cell cycle phases, or after a treatment of interest can be
done through Chip assay.
Temporary cross-linking of protein and associated chromatin is
done in live cells or tissues with the help of formaldehyde or
UV.
Then these are subjected to shearing by using enzymatic
digestion or sonication to obtain approx. 300-1000 bp DNA
fragments.
By using a specific antibody, the protein of interest with any
attached fragment of DNA is imunoprecipitated from the debris of
cells.
Then, the reversal of cross-linking and purification of DNA
fragments takes place.
The eluted DNA quantity can be assessed by qRT-PCR with a
primer that mimic the genomic locus of interest.
An amplification of DNA indicates an enhancement in binding of
the protein of interest.