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Methanol

甲醇

Company: Sigma-Aldrich
Catalog#: 322415
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Expression and Ni-NTA-Agarose Purification of Recombinant Hepatitis C Virus E2 Ectodomain Produced in a Baculovirus Expression System
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Date:
2018-10-05
[Abstract]  In this protocol, we describe the production and purification of the ectodomain of the E2661 envelope protein (amino acids 384-661) of the Hepatitis C virus, which plays a fundamental role in the entry of the virus into the host cell. This protein has been expressed in both prokaryotic and eukaryotic systems but in small quantities or without native protein characteristics. In our case, we use the Baculovirus expression system in insect cells. E2661 is secreted into the extracellular medium and purified by means of affinity chromatography a Ni-NTA-column because the protein has a tag of six histidines at its amino terminal end. The purified protein possesses a native-like conformation and it is produced in large quantities, around 5-6 mg per liter. [摘要]  在该协议中,我们描述了丙型肝炎病毒的E2 661 包膜蛋白(氨基酸384-661)的胞外域的产生和纯化,其在病毒进入中起基础作用。 进入宿主细胞。 该蛋白质已经在原核和真核系统中表达,但是少量或没有天然蛋白质特征。 在我们的例子中,我们在昆虫细胞中使用杆状病毒表达系统。 E2 661 被分泌到细胞外培养基中并通过亲和层析Ni-NTA-柱纯化,因为该蛋白质在其氨基末端具有六个组氨酸的标签。 纯化的蛋白质具有天然样构象,并且大量生产,每升约5-6mg。
【背景】丙型肝炎病毒(HCV)是全世界慢性肝炎,肝硬化和肝细胞癌的主要原因(Major et al。,2001; Alter,2006)。此时,没有HCV疫苗,抗病毒药物用于治疗HCV感染(Imran et al。,2014)。然而,治疗费用昂贵且不是100%有效(Kohli et al。,2014)。 HCV包膜糖蛋白E2负责与细胞受体的相互作用,因此它是研究病毒感染周期的第一步的主要候选者。由于糖基化和聚集,先前的表达系统产生低水平的异质蛋白质,并且难以区分经历生产性和非生产性折叠的分子(Flint ...

Isolation of Intact Vacuoles from Petunia Petals and Extraction of Sequestered Glycosylated Phenylpropanoid Compounds
Author:
Date:
2018-07-05
[Abstract]  Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles (Paris et al., 1996; Olbrich et al., 2007). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids (Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation ... [摘要]  植物液泡是植物细胞中最大的隔室,占细胞体积的80%以上。各种蛋白质,糖,色素和其他代谢物存储在这些细胞器中(Paris et al。,1996; Olbrich et al。,2007)。花产生多种特殊代谢物,其中一些是该器官特有的,如授粉综合征的成分, ie ,气味挥发物和黄酮类化合物(Hoballah et al。, 2007; Cna'ani et al。,2015)。为了研究存储在花液泡中的化合物,必须将该隔室与细胞的其余部分分开。为了能够分离液泡,首先通过将刺穿的花冠与纤维素酶和macrozyme酶一起孵育来产生原生质体。在过滤和几个离心步骤后,通过显微镜观察显示原生质体与碎片和受损/破裂的原生质体分离。裂解浓缩的原生质体,并通过Ficoll梯度离心提取液泡。 Vacuoles用于隔离代谢物的定量GC-MS分析。这种方法使我们能够将空泡识别为糖基化挥发性苯丙酸类的亚细胞聚集位点,并假设共轭气味化合物在通向顶空的途径中被隔离(Cna'ani et al。,2017) 。

【背景】植物空泡占植物细胞中细胞体积的80%。这些细胞器对植物生长和发育至关重要,在整个植物的生命中具有不同的功能。 ...

Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry
Author:
Date:
2018-07-05
[Abstract]  Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure ... [摘要]  来自产后啮齿动物的小脑颗粒神经元(CGN)已被广泛用作研究神经元发育,生理学和病理学的模型。 CGN体外培养维持成熟小脑颗粒细胞在体内显示的相同特征,包括发育致密的神经炎网络,神经元活动,神经递质释放和神经元的表达 蛋白质标记。 此外,CGN代表了梭菌神经毒素(CNT)研究的便利模型,最着名的是破伤风和肉毒杆菌神经毒素,因为它们大量表达CNT受体和神经元内基质, ie ,可溶性N-乙基马来酰亚胺 - 敏感因子激活蛋白受体(SNARE蛋白)。 在这里,我们描述了从出生后大鼠/小鼠获得高纯度CGN培养物的方案和用CNT中毒的简便方法。 我们还说明了评估CNT活性及其抑制的方便方法。

【背景】梭菌神经毒素(CNT)的大家族由破伤风神经毒素(TeNT)和肉毒杆菌神经毒素(BoNT)的多种变体形成,它们分别是破伤风和肉毒中毒的神经麻痹毒素(Schiavo et al。,2000; Johnson和Montecucco,2008; Rossetto et al。,2014)。 TeNT,七种BoNT血清型(BoNT / A至/ G)及其许多亚型是金属蛋白酶,通过切割SNARE蛋白(可溶性N-乙基马来酰亚胺敏感因子激活蛋白受体),三种必需蛋白质来阻断神经递质的释放而引起神经麻痹。控制突触小泡与突触前质膜的融合(Rossetto et al。,2014; ...

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