Chromatin immunoprecipitation (ChIP) is a deductive in vivo method that allows the investigation of any chromatin component in its natural context. This method can be used to analyze the in vivo distribution of any factor and relate this to the activity of a particular gene or locus. Recent applications of the ChIP technology have also allowed the investigation of binding profiles of specific families of factors and core chromatin modifications on a genome wide scale. Prior to immunoprecipitation of fixed chromatin it is absolutely necessary to test the antibody to check its compatibility with crosslinked material and certain detergents. The criterion to calculate the amount of cells to be grown is that one needs at least 20-30 microgram DNA per immunoprecipitation. When the sequence of the target region of the protein of interest is known, the immunopurified DNA can be directly used as template for a semiquantitative PCR using primer pairs that span the putative binding sites. In this case, amplification is obtained if protein binding occurs, otherwise no amplification will be obtained.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)