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Chromatin structure plays a fundamental role in regulating processes that take place on the DNA, such as transcription, replication, and recombination, all of which occur on nucleosomal templates. Therefore, mapping the position of histones selectively modified and histone variants or non-histone components of chromatin in specific DNA sequences, can provide valuable insights into how these proteins (and their modifications) function in a chromatin context. Chromatin immunoprecipitation (ChIP) is a powerful tool for identifying proteins, including histone proteins and non-histone proteins, associated with specific regions of the genome by using specific antibodies that recognize a specific protein or a specific modification of a protein. The method is based on the principle that formaldehyde reacts with primary amines located on amino acids and the bases on DNA or RNA molecules, forming a covalent crosslink between the specific protein to the DNA on which they are situated. Once the DNA binding proteins are crosslinked, the cells are lysed and the crude cell extracts are sonicated to shear the DNA to a smaller size. The protein:DNA complex is immunoprecipitated using an antibody against the protein of interest. The DNA protein cross-links are reversed by heating and the proteins removed by treatment with proteinase K. The DNA portion of the complex is then purified and identified by PCR using specific primers to the suspected binding region. Successful chromatin immunoprecipitation requires an effective, specific, and high quality antibody. Not all antibodies work in ChIP as the antigen epitope may be blocked or altered once cross-linked. Sigma’s Imprint® brand of antibodies are validated in ChIP application and lot tested each time for successful chromatin immunoprecipitation experiment.
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