title: Chromatin immunoprecipitation analysis of Xenopus embryos.
abstract: Chromatin immunoprecipitation (ChIP) is a powerful technique to study epigenetic regulation and transcription factor binding events in the nucleus. It is based on immune-affinity capture of epitopes that have been cross-linked to genomic DNA in vivo. A readout of the extent to which the epitope is associated with particular genomic regions can be obtained by quantitative PCR (ChIP-qPCR), microarray hybridization (ChIP-chip), or deep sequencing (ChIP-seq). ChIP can be used for molecular and quantitative analyses of histone modifications, transcription factors, and elongating RNA polymerase II at specific loci. It can also be applied to assess the cellular state of transcriptional activation or repression as a predictor of the cells' capabilities and potential. Another possibility is to employ ChIP to characterize genomes, as histone modifications and binding events occur at specific and highly characteristic genomic elements and locations. This chapter provides a step-by-step protocol of ChIP using early Xenopus embryos and discusses potential pitfalls and other issues relevant for successful probing of protein-genome interactions by ChIP-qPCR and ChIP-seq.
authors.affiliation: Department of Molecular Biology, Nijmegen Center of Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Akkers
authors.forename: Ulrike G
authors.initials: UG
authors.lastname: Jacobi
authors.forename: Gert Jan C
authors.initials: GJ
authors.lastname: Veenstra
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D002843:Chromatin / Q000235:genetics / Q000302:isolation & purification; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000166:cytology*; D005326:Fetal Proteins / Q000235:genetics; D056426:Genetic Loci; D059014:High-Throughput Nucleotide Sequencing; D006657:Histones / Q000378:metabolism; D008745:Methylation; D020411:Oligonucleotide Array Sequence Analysis; D011499:Protein Processing, Post-Translational; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013010:Sonication; D020825:T-Box Domain Proteins / Q000235:genetics; D014981:Xenopus / Q000235:genetics*; D029867:Xenopus Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000098908:Brachyury Protein
chemical_list: D000906:Antibodies; D002843:Chromatin; D004247:DNA; D017931:DNA Primers; D005326:Fetal Proteins; D006657:Histones; D020825:T-Box Domain Proteins; D029867:Xenopus Proteins; D000098908:Brachyury Protein