Dahl John Arne, Collas Philippe
Department of Biochemistry, Faculty of Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo 0317, Norway.
Nat Protoc. 2008;3(6):1032-45. doi: 10.1038/nprot.2008.68.
Interactions of proteins with DNA mediate many critical nuclear functions. Chromatin immunoprecipitation (ChIP) is a robust technique for studying protein-DNA interactions. Current ChIP assays, however, either require large cell numbers, which prevent their application to rare cell samples or small-tissue biopsies, or involve lengthy procedures. We describe here a 1-day micro ChIP (microChIP) protocol suitable for up to eight parallel histone and/or transcription factor immunoprecipitations from a single batch of 1,000 cells. MicroChIP technique is also suitable for monitoring the association of one protein with multiple genomic sites in 100 cells. Alterations in cross-linking and chromatin preparation steps also make microChIP applicable to approximately 1-mm(3) fresh- or frozen-tissue biopsies. From cell fixation to PCR-ready DNA, the procedure takes approximately 8 h for 16 ChIPs.
蛋白质与DNA的相互作用介导了许多关键的核功能。染色质免疫沉淀(ChIP)是研究蛋白质-DNA相互作用的一项可靠技术。然而,目前的ChIP分析方法要么需要大量细胞,这使得它们无法应用于稀有细胞样本或小组织活检,要么涉及冗长的程序。我们在此描述一种1天的微量ChIP(microChIP)方案,适用于从一批1000个细胞中进行多达8个平行的组蛋白和/或转录因子免疫沉淀。MicroChIP技术也适用于监测100个细胞中一种蛋白质与多个基因组位点的关联。交联和染色质制备步骤的改变也使microChIP适用于约1立方毫米的新鲜或冷冻组织活检。从细胞固定到可用于PCR的DNA,该程序对16次ChIP而言大约需要8小时。