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N-Lauroylsarcosine sodium salt

N-月桂酰肌氨酸钠盐

Company: Sigma-Aldrich
Catalog#: L9150
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Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
Author:
Date:
2017-06-05
[Abstract]  The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart. [摘要]  DNA梳理方法允许在沿着硅烷涂覆的玻璃盖玻片拉伸的单个DNA分子的水平上分析DNA复制。在DNA提取前,进行的DNA合成用胸苷的卤化类似物标记。使用特异性抗体通过免疫荧光可视化复制轨迹。与生物化学和基于NGS的方法不同,DNA梳理提供了DNA复制谱中细胞间细胞变化的独特信息,包括引发和延长。最后,该测定可用于监测DNA损伤对叉进展,停止和重新启动的影响。

背景 在称为复制起点的真核染色体上的数千个位点处启动DNA合成。原始激活遵循由检查点激酶和染色质的表观遗传修饰(Prioleau和MacAlpine,2016)控制的定义良好的复制计时程序。复制叉在正常S阶段经常停顿。叉停止是由多个事件引起的,例如DNA损伤,紧密结合的蛋白质复合物和高表达基因的转录(Tourriere和Pasero 2007; Zeman and Cimprich,2013)。真核生物已经制定了不同的策略来应对这种复制压力,包括修复机制来重新启动捕获的叉子和激活休眠复制起源以抢救终末抓捕的叉。
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Heterologous Expression and Purification of Catalytic Domain of CESA1 from Arabidopsis thaliana
Author:
Date:
2016-10-20
[Abstract]  Heterologous expression of plant cellulose synthase (CESA) and its purification has remained a challenge for decades impeding detailed biophysical, biochemical and structural characterization of this key enzyme. An in-depth knowledge of structure and function of CESA proteins would enable us to better understand the hierarchical structure of the plant cell wall. Here, we report a detailed, and reproducible method of purification of catalytic domain of CESA1 from Arabidopsis thaliana that was recombinantly expressed in Escherichia coli. The method relies on a two stage purification procedure to obtain the catalytic domain in monomer and trimer forms. The biochemical and biophysical data including low resolution structures of the protein have been published (Vandavasi et ... [摘要]  植物纤维素合酶(CESA)的异源表达及其纯化在数十年来一直是阻碍该关键酶的详细生物物理,生物化学和结构表征的挑战。对CESA蛋白的结构和功能的深入了解将使我们能够更好地理解植物细胞壁的层次结构。在这里,我们报告了在大肠杆菌中重组表达的来自拟南芥的CESA1的催化结构域的详细的和可重复的纯化方法。该方法依赖于两阶段纯化方法以获得单体和三聚体形式的催化结构域。已经公开了包括蛋白质的低分辨率结构的生物化学和生物物理数据(Vandavasi等人,2016)。目前,这种蛋白质的结晶研究正在进行中。

[Backg 圆形] 纤维素是植物细胞壁最重要的结构组分,地球最大的生物可再生材料来源,但它的植物合成机制了解很少。植物纤维素合成复合物(CSC),也称为"玫瑰花结",因为其在电子显微镜图像中的六聚体外观,是一个大的多亚基跨膜蛋白复合物负责纤维素链的合成和它们组装成微原纤维。 ...

Semi-denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE)
Author:
Date:
2014-11-20
[Abstract]  Pathological proteins in neurodegenerative diseases suffer a conformational change to a misfolded amyloid state. Such pathological event leads to the aggregation of these proteins that indefinitely propagates as an altered form of itself, and harbor prion-like properties (Wickner, 1994; Prusiner, 2012). In addition to diseases, prions can also have beneficial adaptive roles in lower eukaryotes (in fungi and yeast) (Eaglestone et al., 1999; True et al., 2004; Coustou et al., 1999). Besides separating polymers from their precursor soluble monomers, another particular difficulty of the study of amyloid proteins is to resolve the heterogeneity of the aggregates, since these usually exhibit a variable degree of polymorphism. Semi-denaturating detergent agarose gel ... [摘要]  神经退行性疾病中的病理蛋白质遭受错误折叠的淀粉样蛋白状态的构象变化。这种病理性事件导致这些蛋白质的聚集,其作为其自身的改变形式无限地繁殖,并且具有朊病毒样特性(Wickner,1994; Prusiner,2012)。除了疾病,朊病毒还可以在低等真核生物(真菌和酵母)中具有有益的适应性作用(Eaglestone等人,1999; True等人,2004; Coustou等人,1999)。除了从其前体可溶性单体中分离聚合物之外,研究淀粉样蛋白的另一个特别困难是解决聚集体的异质性,因为这些通常表现出不同程度的多态性。半变性洗涤剂琼脂糖凝胶电泳(SDD-AGE)是一种利用朊病毒和朊病毒样聚合物的性质来高度抵抗SDS洗涤剂的溶解的技术,以及琼脂糖的大孔径,其允许分辨率的高分子量复合物。在该方法中,我们详细描述了该技术如何用于表征细菌和酵母中的异质聚集(Gasset-Rosa等人,2014; Molina-García和Giraldo,2014)应用于研究通过遗传操作变得易于聚集的蛋白质的聚集模式。

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