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Imidazole

咪唑

Company: Sigma-Aldrich
Catalog#: I5513
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Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation
Author:
Date:
2020-05-20
[Abstract]  Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the mechanism of histone transport and also the potential roles of importins as histone chaperones and in the assembly of nucleosomes. Here we report a simple and reproducible sedimentation-velocity based method to determine the molecular sizes of importin-histone complexes using analytical ultracentrifugation. The method does not use any reporter tags or interaction with column resin thereby analyzing the interactions of the native proteins. [摘要]  [摘要] 组蛋白构成核小体的蛋白质成分。尽管其尺寸很小,但组蛋白不会通过核孔复合物扩散。取而代之的是,它们单独或与组蛋白分子伴侣复合地被重要蛋白转运至细胞核。确定importin-histone复合物的分子大小是理解组蛋白转运机制的关键,也是importins作为组蛋白伴侣和在核小体组装中的潜在作用的关键。在这里,我们报告了一种简单且可重现的沉降速度为基础的方法,该方法使用分析超速离心法来确定importin-histone配合物的分子大小。该方法不使用任何报告子标签或与色谱柱树脂的相互作用,从而分析了天然蛋白质的相互作用。

[背景] 核小体是真核染色质的最基本的结构和功能单元。组蛋白H2A,H2B,H3和H 4是核小体的蛋白质成分。每个核包括147个碱基的DNA wrapp的对编绕Ñ H3-H4四聚体和H2A-H2B二聚体的两个拷贝(Luger的等人,1997年一)。像细胞中的其他蛋白质一样,组蛋白在细胞质中合成。然而,核小体组装在核中。尽管它们的小尺寸(单体是10-15 kDa)的,组蛋白不通过核孔复合物扩散,而是可以单独使用或在复合物与由组蛋白importins伴侣输送要么(约翰逊-SAL IBA 等人,2000 ; Baake 等等人,2001;Mosammaparast 等人,2001,2002a和2002b;Muhlhausser ...

Laminarin Quantification in Microalgae with Enzymes from Marine Microbes
Author:
Date:
2018-04-20
[Abstract]  The marine beta-glucan laminarin is an abundant storage polysaccharide in microalgae. High production rates and rapid digestion by heterotrophic bacteria turn laminarin into an ideal carbon and energy source, and it is therefore a key player in the marine carbon cycle. As a main storage glucan laminarin also plays a central role in the energy metabolism of the microalgae (Percival and Ross, 1951; Myklestad, 1974; Painter, 1983). We take advantage of enzymes that digest laminarin selectively and can thereby quantify only this polysaccharide in environmental samples. These enzymes hydrolyze laminarin into glucose and oligosaccharides, which are measured with a standard reducing sugar assay to obtain the laminarin concentration. Prior to this assay, the three enzymes need to be produced via ... [摘要]  海洋β-葡聚糖昆布多糖是微藻中丰富的储存多糖。高生产率和异养细菌的快速消化将昆布多糖转化为理想的碳源和能源,因此它是海洋碳循环的关键参与者。作为主要的储存葡聚糖昆布多糖也在微藻的能量代谢中发挥核心作用(Percival and Ross,1951; Myklestad,1974; Painter,1983)。我们利用可以选择性消化昆布多糖的酶,从而可以对环境样品中的这种多糖进行定量。这些酶将昆布多糖水解成葡萄糖和寡糖,用标准的还原糖测定法测定得到昆布多糖浓度。在此测定之前,需要通过异源表达和纯化产生三种酶。该测定可用于监测环境微藻中的昆布多糖浓度,其通过过滤从海水中浓缩,或用来自藻类实验室培养物的样品中浓缩。

【背景】海洋多糖在海洋碳循环中起着重要作用,是浮游植物生理学的重要组成部分,但受到严重影响。几十年来,农业食品工业一直使用基于酶分析的即用试剂盒来分析各种不同的多糖(Whitaker,1974)。这些快速,稳健和特异性的基于酶的方法评估源自陆地植物即淀粉的多糖,因为它们广泛用于食品,饲料和其他工业应用中(Brunt等人, ,1998)。然而,海洋多糖的类似测定仍然缺乏。受到使用酶在藻类中进行多糖定量的想法的启发,我们开发了一种基于酶的方法来量化在硅藻和其他微藻中生态相关的β-葡聚糖昆布氨酸,也称为菊科金刚烷。

这种应用的三种糖苷水解酶(GH)来自福尔摩沙(Formosa)。并且它们的特征如下:FbGH30是GH30家族的外切型β-1,6-葡聚糖酶,特别是水解与昆布多糖骨架连接的β-1,6-连接的葡萄糖单体分支;并且FaGH17A和FbGH17A是GH家族17的两种内作用β-1,3-葡聚糖酶,其特异性地作用于β-1,3-连接的昆布多糖主链上(Becker等人,2017年, ...

Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor
Author:
Date:
2017-06-20
[Abstract]  Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide. Individual receptor molecules are then monitored over time by single-molecule total internal reflection fluorescence microscopy, revealing spontaneous transitions between inactive and active-like conformations. The assay provides information on the equilibrium distribution of inactive and active receptor conformations and ... [摘要]  通过激动剂配体激活G蛋白偶联受体(GPCR)是通过从无活性受体构象向活性受体构象的转变来介导的。我们描述了一种新颖的单分子测定法,可以实时监测单个GPCR分子中的激活连锁构象转换。受体在跨膜螺旋6末端用Cy3荧光探针进行位点特异性标记,并在连接到显微镜载玻片的磷脂纳米圆盘中重构。然后通过单分子全内反射荧光显微镜随时间监测单个受体分子,显示无活性和活性样构象之间的自发转变。该测定提供关于无活性和活性受体构象的平衡分布以及构象交换的速率常数的信息。实验可以在不存在配体的情况下进行,显示负责基础信号传导活动的自发构象过渡,或者存在激动剂或反向激动剂配体,揭示配体如何改变受体的动力学刺激或抑制信号传导活性。所得到的机械信息对于改进的GPCR靶向药物的设计是有用的。单分子测定法在β2肾上腺素能受体的背景下进行了描述,但可扩展到多种GPCRs。
【背景】GPCR介导本地和远距离的细胞通讯,特别是内分泌系统。例如,细胞对激素如肾上腺素的反应是通过肾上腺素能受体介导的,其中β2肾上腺素能受体(β2AR)是突出的成员。 ...

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