Question

What is Chromatin Immunoprecipitation (ChIP) and how does it work?

What is Chromatin Immunoprecipitation (ChIP) and how does it work?

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Ans.

  • 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) -

  • 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.
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