| Chromatin Immunoprecipitation (ChIP) to Assess Histone Marks in Auxin-treated Arabidopsis thaliana Inflorescence Tissue
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Author:
Date:
2020-12-05
[Abstract] Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) or high-throughput sequencing (ChIP-seq) has become the gold standard for the identification of binding sites of DNA binding proteins and the localization of histone modification on a locus-specific or genome-wide scale, respectively. ChIP experiments can be divided into seven critical steps: (A) sample collection, (B) crosslinking of proteins to DNA, (C) nuclear extraction, (D) chromatin isolation and fragmentation by sonication, (E) immunoprecipitation of histone marks by appropriate antibodies, (F) DNA recovery, and (G) identification of precipitated protein-associated DNA by qPCR or high-throughput sequencing. Here, we describe a time-efficient protocol that can be used for ChIP-qPCR experiments to study the ...
[摘要] [摘要]染色质免疫沉淀与定量PCR（ChIP -qPCR）或高通量测序（ChIP-seq ）结合已成为鉴定DNA结合蛋白结合位点和在特定基因座上定位组蛋白修饰的金标准。或全基因组规模。ChIP实验可分为七个关键步骤：（A）样品收集，（B）蛋白质与DNA交联，（C）核提取，（D）染色质分离和f 超声处理的碎片化；（E）通过适当的抗体对组蛋白标记的免疫沉淀；（F）DNA的回收；（G）通过qPCR或高通量测序鉴定沉淀的蛋白质相关DNA。在这里，我们描述了一种可用于ChIP -qPCR实验的省时协议，以研究模型植物拟南芥幼花序中组蛋白修饰的定位。
[背景]真核基因组中的染色体中，其与组蛋白DNA结合形成染色质组织的。组蛋白与DNA之间的紧密相互作用阻碍了DNA与其他因素的可及性。因此，组蛋白相对于重要调控DNA序列的位置和组蛋白-DNA接触的强度可以隐藏或暴露提供另一层基因调控的基因。在染色质中，组蛋白和DNA均可被化学修饰（Zhou等，2010 ；Schübeler ，2015）。根据修饰的物理性质，染色质状态可以阻止或增强基础基因的转录（Kouzarides ，2007； Yang等，2014； Wu等，2015）。在植物中，染色质的表观遗传状态已被证明是响应发育或环境刺激的基因表达的关键决定因素（Yang等人，2014 ; Wu等人，2015 ; ...
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| Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures
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Author:
Date:
2020-07-05
[Abstract] MYC family members, MYC, MYCN, and MYCL, are oncogenic transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. While MYC is amplified and/or overexpressed across a variety of tissue types, MYCN is often overexpressed in tumors of the nervous system (neuroblastoma and medulloblastoma) or with neuroendocrine features (neuroendocrine prostate cancer). Given recent reports that MYCN expression is also deregulated in a variety of non-neuronal tissue types, we investigated whether MYCN was also deregulated in triple-negative breast cancer (TNBC). In contrast to previous individual immuno-fluorescence (IF) stains against higher expressing MYC family isoform protein, we developed an IF stain to ...
[摘要] [摘要] MYC家族成员MYC、MYCN和MYCL是一类致癌转录因子，它们调节与正常发育、细胞生长、增殖、代谢和生存有关的基因的表达。虽然MYC在多种组织类型中扩增和/或过度表达，但MYCN通常在神经系统肿瘤（神经母细胞瘤和髓母细胞瘤）或具有神经内分泌特征（神经内分泌前列腺癌）中过度表达。鉴于最近的报道，MYCN在多种非神经元组织中的表达也被解除了调控，我们研究了MYCN在三阴性乳腺癌（TNBC）中是否也被解除了调控。与以往针对高表达MYC家族亚型蛋白的个体免疫荧光（IF）染色不同，我们开发了一种IF染色法来同时检测同一肿瘤细胞群中MYCN-和MYC表达细胞。我们的方法允许检测低水平的MYCN和MYC表达，并且可以与额外的蛋白质探针复合。在此，我们利用酪酰胺信号放大（TSA），提出了两种检测MYCN和MYC的方案，用于在福尔马林固定石蜡包埋（FFPE）肿瘤切片和生长后原位固定的细胞系中检测MYCN和MYC。
[背景] ...
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| Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis
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Author:
Date:
2018-10-05
[Abstract] Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes a method to efficiently fractionate cells combined with a mechanical (i.e., sonication) or an enzymatic treatment (i.e., benzonase) that facilitates analysis of chromatin-bound protein extracts by Western blot analysis or by protein pull-down assays. This approach can be valuable to enrich a particular protein within a particular subcellular fraction either to study specific post-translational modification patterns or to ...
[摘要] 在不同细胞区室之间穿梭蛋白质控制它们的蛋白质稳态，并且在某些情况下可以有助于调节它们的活性。 染色质结合蛋白（例如转录因子）的生化分析通常是困难的，因为它们的产率低并且由于核酸的干扰。 该协议描述了一种有效分离细胞的方法，结合机械（即，超声处理）或酶处理（即，benzonase），有助于分析染色质结合蛋白提取物 通过蛋白质印迹分析或蛋白质下拉分析。 该方法对于富集特定亚细胞级分内的特定蛋白质以研究特定的翻译后修饰模式或鉴定特定的蛋白质 - 蛋白质相互作用可能是有价值的。
【背景】许多染色质结合蛋白的活性和翻译后调节研究很少，因为在分离它们进行生化分析时存在技术困难。这甚至是转录因子的情况，例如基本的螺旋 - 环 - 螺旋（bHLH）转录因子，其通常在组织或细胞模型中具有稀缺的时间和空间表达模式（Dennis 等。，2018）。当生物材料的量成为研究分子途径的障碍时，协议细化有助于解除技术限制（Gillotin和Guillemot，2016）。在我们最近的研究中，我们努力了解神经元bHLH转录因子Ascl1的蛋白水解是如何在神经元分化的细胞模型中调节的（Gillotin et ...
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