Bhatia Shama, Matthews Jason, Wells Peter G
Department of Pharmaceutical Sciences and Centre for Pharmaceutical Oncology, University of Toronto, Toronto, ON, Canada.
Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2019;1965:389-403. doi: 10.1007/978-1-4939-9182-2_25.
Chromatin immunoprecipitation (ChIP) is widely used to measure protein-DNA interactions. This protocol outlines a ChIP method used to identify the association of a protein or protein modification (such as a specific histone modification-methylation, acetylation, etc.) of interest with a specific DNA sequence in a target gene in fetal mouse brains on gestational day (GD) 17. Briefly, DNA and proteins are cross-linked (via formaldehyde), and chromatin is sonicated into fragments between 200 and 1000 base pair (bp) long, with an average length of 500 bp. The DNA-protein complexes are captured using antibodies directed toward the protein or protein modification of interest. These immunoprecipitated complexes are retrieved using agarose beads. The DNA-protein cross-links are reversed (via heat and via presence of high salt concentrations), and the ChIP DNA is purified and measured via a quantitative polymerase chain (qPCR) reaction. The results show the association of histone modifications at unknown sites of specific genes of interest, indicating which epigenetic modifications of specific genes may be responsible for the outcome of interest.
染色质免疫沉淀法（ChIP）被广泛用于测量蛋白质与DNA的相互作用。本方案概述了一种ChIP方法，用于鉴定妊娠第17天的胎鼠大脑中目标基因的特定DNA序列与感兴趣的蛋白质或蛋白质修饰（如特定的组蛋白修饰——甲基化、乙酰化等）之间的关联。简而言之，DNA和蛋白质进行交联（通过甲醛），染色质被超声破碎成长度在200至1000碱基对（bp）之间、平均长度为500 bp的片段。使用针对感兴趣的蛋白质或蛋白质修饰的抗体捕获DNA-蛋白质复合物。这些免疫沉淀的复合物用琼脂糖珠回收。DNA-蛋白质交联被逆转（通过加热和高盐浓度），ChIP DNA被纯化并通过定量聚合酶链反应（qPCR）进行测量。结果显示了特定感兴趣基因未知位点处组蛋白修饰的关联，表明特定基因的哪些表观遗传修饰可能导致感兴趣的结果。
