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Glycerol

甘油

Company: Sigma-Aldrich
Catalog#: G5516
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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 ...

A Method for SUMO Modification of Proteins in vitro
Author:
Date:
2018-10-05
[Abstract]  The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification in vivo is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes known as SUMO Isopeptidases or SENPs. Fortunately, the use of recombinant SUMO enzymes makes it possible to study SUMO modification in vitro. Here, we describe a sensitive method for ... [摘要]  小泛素相关修饰物(SUMO)是一种蛋白质,其翻译后添加到真核细胞中并可逆地从其他蛋白质中去除。 SUMO和SUMO途径的酶从酵母到人类都很保守,SUMO修饰调节了多种基本细胞过程,包括转录,染色质重塑,DNA损伤修复和细胞周期进程。 研究SUMO修饰体内的挑战之一是SUMO修饰蛋白的相对低的稳态水平,部分原因是SUMO去缀合酶(SUMO Isopeptidases或SENPs)的活性。 幸运的是,使用重组SUMO酶可以在体外研究SUMO修饰。 在这里,我们描述了一种灵敏的方法,用于检测目标人类蛋白质的SUMO修饰,使用来自兔网织红细胞和放射性标记的氨基酸的体外转录和翻译系统。
【背景】与其他泛素蛋白家族修饰一样,SUMO修饰通过ATP依赖性酶促级联发生,涉及E1激活酶(人类中的Aos1 / Uba2异二聚体),E2结合酶(Ubc9)和许多E3连接之一的连续活性。酶(Gareau和Lima,2010)。具有SUMO缀合共有位点的蛋白质ΨKxE(Ψ是疏水残基,其后是赖氨酸,任何氨基酸和谷氨酸),可以通过哺乳动物中表达的一种或几种SUMO旁系同源物(包括SUMO1,SUMO2)进行有效修饰。或SUMO3(统称为SUMO2 / 3,因为它们的序列同源性为97%)(Gareau和Lima,2010; Flotho和Melchior,2013)。 ...

Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis
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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