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Trizma ® base

Company: Sigma-Aldrich
Catalog#: T1503
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Transcytosis Assay for Transport of Glycosphingolipids across MDCK-II Cells
Author:
Date:
2018-10-20
[Abstract]  Absorption and secretion of peptide and protein cargoes across single-cell thick mucosal and endothelial barriers occurs by active endocytic and vesicular trafficking that connects one side of the epithelial or endothelial cell (the lumen) with the other (the serosa or blood). Assays that assess this pathway must robustly control for non-specific and passive solute flux through weak or damaged intercellular junctions that seal the epithelial or endothelial cells together. Here we describe an in vitro cell culture Transwell assay for transcytosis of therapeutic peptides linked covalently to various species of the glycosphingolipid GM1. We recently used this assay to develop technology that harnesses endogenous mechanism of lipid sorting across epithelial cell barriers to enable ... [摘要]  单细胞厚粘膜和内皮屏障上的肽和蛋白质货物的吸收和分泌通过活性内吞和囊泡运输发生,其连接上皮细胞或内皮细胞(管腔)的一侧与另一侧(浆膜或血液)。 评估该途径的测定必须通过弱的或受损的细胞间连接强有力地控制非特异性和被动的溶质通量,所述细胞间连接将上皮细胞或内皮细胞密封在一起。 在这里,我们描述了一种体外>细胞培养Transwell测定法,用于与各种鞘糖脂GM1共价连接的治疗性肽的转胞吞作用。 我们最近使用该测定开发了技术,该技术利用跨上皮细胞屏障的脂质分选的内源机制,以实现肽和蛋白质治疗剂的口服递送。

【背景】
大分子穿过覆盖粘膜表面的单细胞厚的上皮屏障和衬在供给心肌和脑的血管的紧密内皮屏障上的运输通过内吞过程发生,该内吞过程将这些极化细胞的一侧与另一侧连接。该过程称为转胞吞作用(Garcia-Castillo et al。>,2017)。通过受体介导的内吞作用和穿过消化道和呼吸道粘膜的囊泡运输的免疫球蛋白的吸收和分泌最着名的是这一过程。对转胞吞作用的兴趣也受到利用该途径在紧密上皮和内皮屏障上递送治疗性肽和蛋白质的潜力的刺激(Thuenauer 等人,>,2017)。

转胞吞作用是一种活跃的(ATP驱动的)过程。在一些情况下,通过细胞间紧密连接在细胞周围被动扩散可以发生大分子跨越紧密上皮和内皮屏障的转运(Fung ...

Expression and Ni-NTA-Agarose Purification of Recombinant Hepatitis C Virus E2 Ectodomain Produced in a Baculovirus Expression System
Author:
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)。 ...

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