Chromatin immunoprecipitation (ChIP) was recently developed and is a very powerful tool to study in vivo interaction between proteins and DNA. ChIP can be extremely useful in the study of transcription, as it enables the identification of DNA regions that contain a transcription factor binding site or that are susceptible to histone modifications such as methylation or acetylation. It is, however, technically highly demanding and requires efficient antibodies, particularly when analyzing transcription factors as opposed to more abundant histone proteins.
The technique relies on fixing a protein of interest on its DNA target in living cells, in order to isolate this specific complex, followed by PCR amplification to detect the target DNA. Intact cells are fixed using formaldehyde, which cross-links protein to the DNA and therefore preserves specific protein-DNA complexes. The DNA is then sheared into small pieces using ultrasound. The complex between the protein of interest and its target DNA is immunoprecipitated with an antibody specific to the protein, using protein G agarose or magnetic beads. The DNA is extracted using a proteinase K and phenol method, in order to reverse the cross-linking and remove the proteins and antibodies. A PCR based method is then used to detect the presence or absence of the target DNA in the immunoprecipitate. Due to the very limited amount of DNA precipitated, particularly when looking for transcription factor binding sites, a real-time method is highly recommended and SYBR® Green assays perform very well (Wang et al., 2004, Potratz et al., 2005).
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