| Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells
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Author:
Date:
2017-03-20
[Abstract] Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome integrity. The extent of association with chromatin changes rapidly in response to stresses, such as immune activation, oxidative stress, or viral infection, resulting in downstream effects on chromatin conformation and transcription of target genes. To elucidate changes in the composition of proteins associated with chromatin under different conditions, we adapted existing protocols to isolate nuclei and fractionate cellular chromatin using a ...
[摘要] 核小体是细胞染色质的核心单元，由包裹在组蛋白的八聚体周围的147个碱基对（bp）的DNA组成。蛋白质如染色质重组体，转录因子和DNA修复蛋白质与染色质动态相互作用以调控DNA的接触，控制基因转录和维持基因组完整性。与染色质结合的程度响应于诸如免疫激活，氧化应激或病毒感染等应激而迅速变化，导致对染色质构象和靶基因转录的下游作用。为了阐明在不同条件下与染色质相关蛋白质组成的变化，我们调整了现有的方案来分离核，并使用盐浓度的梯度分离细胞染色质。可以通过蛋白质印迹或质谱法评估不同盐部分中特定蛋白质的存在，从而了解与染色质相关的程度。
背景 由于与DNA或组蛋白的电荷相互作用，许多染色质相关蛋白在低盐条件下是不溶的。由于盐破坏了基于电荷的蛋白质DNA和蛋白质 - 蛋白质的相互作用，染色质相关蛋白质随着NaCl浓度的增加而变得更加可溶（Teves和Henikoff，2012）。与DNA强烈结合的蛋白质预期用高盐洗脱，而松散结合的蛋白质（例如转录因子）将用低盐洗脱。我们特别关心病毒感染如何改变与细胞染色质相关的因素的组成。核复制病毒，例如腺病毒，单纯疱疹病毒和爱泼斯坦 - 巴尔病毒，可以显着改变感染期间宿主染色质的出现（Avgousti等人，2016; Lam等人， ，2010; Simpson-Holley等人，2005; ...
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| Procedure for Rhamnolipids Quantification Using Methylene-blue
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Author:
Date:
2016-04-05
[Abstract] Rhamnolipids produced by Pseudomonas aeruginosa (P. aeruginosa) represent a group of biosurfactants with various applications (e.g., bioremediation of oil spills, cosmetics, detergents and cleaners). The commonly used colorimetric methods for rhamnolipid quantification, including anthrone, phenol−sulfuric acid and orcinol based quantification (Helbert and Brown, 1957; Chandrasekaran and BeMiller, 1980), are laborious and operationally hazardous because of the strong acid/chemical emanation which can cause deterioration of instruments measurements (e.g., spectrophotometer). Therefore, the methylene-blue-based analysis appears as a promising alternative to safely quantify whole rhamnolipid molecules based on chemical complexation reaction (Pinzon and Ju, ...
[摘要] 由绿脓杆菌（铜绿假单胞菌）产生的鼠李糖脂代表一组具有各种应用的生物表面活性剂（例如生物修复的溢油，化妆品，洗涤剂和清洁剂）。常用的鼠李糖脂定量的比色方法，包括蒽酮，苯酚 - 硫酸和基于苔藓醇的定量（Helbert和Brown，1957; Chandrasekaran和BeMiller，1980）是费力和操作上危险的，因为强酸/化学散发仪器测量的恶化（例如分光光度计）。因此，基于亚甲蓝的分析显示为基于化学络合反应安全定量整个鼠李糖脂分子的有希望的替代物（Pinzon和Ju，2009）。事实上，亚甲基蓝和鼠李糖脂在水 - 氯仿相系统中形成复合物。将鼠李糖脂 - 亚甲基蓝复合物分配到氯仿相中，其将形成蓝色，其可以在638nm下定量以推断鼠李糖脂浓度。在这里，我们描述基于亚甲基蓝的鼠李糖脂定量程序的变体，允许在标准96孔塑料微量滴定板相反的原始亚甲蓝程序，由于氯仿化学属性需要特定和昂贵的微孔板分光光度定量。
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