Chromatin Immunoprecipitation (ChIP)
Many DNA-binding proteins, such as transcription factors, bind to specific sequences of nucleotides in, for example, promoters and enhancers of genes. View some examples:
The binding of protein to DNA is done by noncovalent forces and is easily reversible. In fact, as conditions in a cell change, there is a dynamic coming and going of DNA-binding proteins throughout the genome.
The identification of a specific site in DNA bound by a particular protein at a particular time can be discovered by the technique of chromatin immunoprecipitation (ChIP).
- Harvest your cell population at the desired time. Some examples:
- Treat the cells with formaldehyde (HCHO) which creates covalent bonds between the proteins and nucleotides to which they have been bound noncovalently.
- Break open the cells releasing their contents.
- Use ultrasound to break the DNA into fragments averaging about 500 bp long.
- Add an antibody that specifically binds to the protein you are interested in.
- Add beads coated with Protein A or Protein S — both proteins that bind to any antibody.
- Centrifuge down the complexes of bead—antibody—target protein—DNA.
- Heat the complexes to break the covalent crosslinks between the target protein and the DNA.
- Digest the protein with a protease leaving purified DNA fragments.
- Perform PCR on the fragments.
- Use any of several methods to identify the amplified DNA, for example,
19 August 2012