Posé David, Yant Levi
Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Instituto de Hortofruticultura Subtropical y Mediterránea, Universidad de Málaga-Consejo Superior de Investigaciones Científicas, 29071, Málaga, Spain.
Department of Cell and Development Biology, John Innes Centre, Norwich Research park, Norwich, NR4 7UH, UK.
Methods Mol Biol. 2016;1363:25-35. doi: 10.1007/978-1-4939-3115-6_3.
Chromatin immunoprecipitation (ChIP) allows the precise identification of genomic loci that physically interact with a protein of interest, whether that protein is a transcription factor, a core polymerase, a histone, or other chromatin-associated protein. In short, tissue is first cross-linked to freeze a population of DNA-protein interactions at a stage of interest. Chromatin is then extracted, fragmented, and incubated with a specific antibody against the protein of interest. Next, the resultant DNA-protein complexes are immunoprecipitated and captured using beads that bind to the antibody constant region. Samples are finally reverse cross-linked to separate the bound fragments and the DNA is purified. This DNA is analyzed by quantitative PCR for enrichment of genomic regions expected to be bound by the protein under study. The protocol detailed in this chapter has been successfully applied in the identification of target genes for seven transcriptional regulators of diverse classes involved in Arabidopsis thaliana floral transition.
染色质免疫沉淀（ChIP）技术能够精确鉴定与目标蛋白发生物理相互作用的基因组位点，无论该蛋白是转录因子、核心聚合酶、组蛋白还是其他与染色质相关的蛋白。简而言之，首先对组织进行交联，以在感兴趣的阶段冻结一群DNA - 蛋白质相互作用。然后提取染色质，进行片段化，并与针对目标蛋白的特异性抗体一起孵育。接下来，使用与抗体恒定区结合的珠子对所得的DNA - 蛋白质复合物进行免疫沉淀和捕获。最后对样品进行反向交联以分离结合的片段，并纯化DNA。通过定量PCR分析该DNA，以富集预期被研究蛋白结合的基因组区域。本章详细介绍的方案已成功应用于鉴定拟南芥花发育转变中涉及的七类不同转录调节因子的靶基因。
