title: Use of in vivo biotinylation for chromatin immunoprecipitation.
abstract: This unit describes a system for expression of biotinylated proteins in mammalian cells in vivo, and its application to chromatin immunoprecipitation (ChIP). The system is based on co-expression of the target protein fused to a short biotin acceptor domain, together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin-avidin interaction in the modified ChIP protocol presented here allows one to employ more stringent washing conditions, resulting in a better signal/noise ratio. Methods for interpreting the data obtained from ChIP samples analyzed by qPCR, and methods for testing the efficiency of biotinylation using a streptavidin gel-shift are also presented. In addition, a complementary method, based on isothermal multiple strand displacement amplification (IMDA) of circular concatemers generated from the DNA fragments obtained after ChIP, is described. This method helps to decrease bias in DNA amplification and is useful for the analysis of complex mixtures of DNA fragments typically generated in miniscale ChIP experiments.
authors.affiliation: CNRS, Université Paris, Villejuif, France.
authors.forename: Arman
authors.initials: A
authors.lastname: Kulyyassov
authors.forename: Muhammad
authors.initials: M
authors.lastname: Shoaib
authors.forename: Vasily
authors.initials: V
authors.lastname: Ogryzko
mesh_terms: D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D002843:Chromatin / Q000737:chemistry; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000737:chemistry; D003627:Data Interpretation, Statistical; D005557:Formaldehyde / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008836:Micrococcal Nuclease / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry*; D013010:Sonication; D019809:Streptavidin / Q000737:chemistry; D014162:Transfection
chemical_list: D002843:Chromatin; D003432:Cross-Linking Reagents; D011993:Recombinant Fusion Proteins; D005557:Formaldehyde; D001710:Biotin; D019809:Streptavidin; D008836:Micrococcal Nuclease