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Hydrochloric acid

盐酸

Company: Sigma-Aldrich
Catalog#: H1758
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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)。 ...

A Quantitative Heterokaryon Assay to Measure the Nucleocytoplasmic Shuttling of Proteins
Author:
Date:
2018-09-05
[Abstract]  Many proteins appear exclusively nuclear at steady-state but in fact shuttle continuously back and forth between the nucleus and the cytoplasm. For example, nuclear RNA-binding proteins (RBPs) often accompany mRNAs to the cytoplasm, where they can regulate subcellular localization, translation and/or decay of their cargos before shuttling back to the nucleus. Nucleocytoplasmic shuttling must be tightly regulated, as mislocalization of several RBPs with prion-like domains such as FUS and TDP-43 causes the cytoplasmic accumulation of solid pathological aggregates that have been implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Traditionally, interspecies heterokaryon assays have been used to determine whether a nuclear ... [摘要]  许多蛋白质在稳态下仅出现核,但事实上在细胞核和细胞质之间连续地来回穿梭。例如,核RNA结合蛋白(RBP)通常伴随mRNA到达细胞质,在那里它们可以在穿梭回到细胞核之前调节其货物的亚细胞定位,翻译和/或腐烂。必须严格调节核质穿梭,因为几种RBP与朊病毒样结构域如FUS和TDP-43的错误定位导致固体病理性聚集体的细胞质积累,这些聚集体与肌萎缩侧索硬化症(ALS)和额颞叶痴呆等神经退行性疾病有关。 (FTD)。传统上,种间异核体分析已被用于确定感兴趣的核蛋白是否穿梭;这些分析是基于来自两个不同物种(例如,小鼠和人类)的供体和受体细胞之间的融合,可以根据不同的染色质染色模式区分,并检测蛋白质的外观。受体核。然而,异核体的鉴定需要经验并且容易出错,这使得难以获得用于定量研究的高质量数据。此外,荧光标记的RBP在供体细胞中的瞬时过表达通常导致其异常的亚细胞定位。在这里,我们提出定量测定,其中表达接近生理水平的eGFP标记的RBP的稳定供体细胞系与表达膜标记物CAAX-mCherry的受体细胞融合,允许容易地鉴定和成像大量高可信度异核体。我们的测定法可用于测量任何感兴趣的核蛋白在不同细胞类型,不同细胞条件下或突变蛋白之间的穿梭活性。

【背景】要了解蛋白质的各种功能,重要的是找出它在细胞内定位的位置。标准的微观和生物化学方法仅在其稳态浓度高于检测阈值时才揭示蛋白质的存在。他们不排除它在短暂地定位的情况下扮演其他重要角色的可能性(Gama-Carvalho和Carmo-Fonseca,2001)。例如,许多RBP在不同的细胞区室中发挥作用,它们伴随着它们的结合mRNA(通常未检测到)并连接真核基因表达的多个步骤(Müller-McNicoll和Neugebauer,2013)。 ...

Mouse Mammary Gland Whole Mount Preparation and Analysis
Author:
Date:
2018-07-05
[Abstract]  The mammary gland undergoes extensive remodeling during pregnancy and is also subject to neoplastic processes both of which result in histological changes of the gland epithelial structure. Since the mammary tree is a complex three-dimensional structure a method is needed that provides an overview of the entire gland. Whole mounts provide this information, are inexpensive and do not require specialized equipment. This protocol describes mammary gland isolation, whole mount preparation and analysis. Mammary gland tissue, which is removed postmortem, is stained with Carmine Alum, a nuclear stain, allowing detection of epithelial structures embedded in the adipose tissue of the mammary fat pad. Stained mammary glands are imaged by light microscopy or embedded and sectioned for histological ... [摘要]  乳腺在怀孕期间经历广泛的重塑并且还经历肿瘤过程,这两者都导致腺体上皮结构的组织学变化。 由于乳树是复杂的三维结构,因此需要一种提供整个腺体概览的方法。 整个安装提供这些信息,价格低廉,不需要专门的设备。 该协议描述了乳腺分离,整体制备和分析。 在死后取出的乳腺组织用胭脂红明矾(一种核染色剂)染色,允许检测嵌入乳腺脂肪垫的脂肪组织中的上皮结构。 通过光学显微镜对染色的乳腺进行成像或嵌入并切片以进行组织学检查。 诸如Image J的图像分析软件可用于量化分支复杂性,上皮结构重塑或增生变化的扩展。

【背景】尽管乳腺的发育在胚胎发育期间开始并且在出生时存在基本的上皮结构,但是上皮乳树在出生后经历广泛的扩张。响应于激素变化,乳腺上皮细胞增殖并侵入乳腺脂肪垫。在怀孕期间,乳腺上皮经历进一步的分化和重塑以准备产奶。随后,这些上皮结构对断奶作出反应。这些重塑过程由激素,生长因子,细胞因子和细胞外基质驱动。除了响应生理过程的重塑之外,乳腺还经历诸如肿瘤转化的病理过程。这种复杂的生物学以及相对容易的分离使得乳腺成为有用的实验模型。分析乳腺生物学或肿瘤转化的实验研究通常使用小鼠模型来量化基因缺失或过表达对乳腺发育,重塑和肿瘤转化的影响。乳腺整个坐骑允许对乳腺的整个3D上皮结构进行常规和疾病过程的常规检查(Plante et al。,2011; Inman et al。, ...

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