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MgCl2

氯化镁六水合物

Company: Sigma-Aldrich
Catalog#: M9272
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In vitro Reconstitution Assays of Arabidopsis 20S Proteasome
Author:
Date:
2021-04-05
[Abstract]  

The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously

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[摘要]  [摘要]大多数细胞蛋白的s的降解通过26S在真核生物蛋白酶。但是,内在无序的蛋白质(IDPs)包含大量的非结构化区域,并且内在地不稳定,因此很容易通过不依赖泛素的20S蛋白酶体降解。越来越多的证据最近显示ň植物境内流离失所者的平衡也可以通过20S蛋白酶控制。但是,由于缺乏用于体外分离20S蛋白酶体和降解测定的系统协议,因此我们对植物中IDP和20S蛋白酶体降解的功能和调控机制的研究和理解一直处于婴儿期与其他生物。在这里,我们通过采用和修改先前公开的方法,对拟南芥中20S蛋白酶体进行体外重组测定的详细方案。在此获得20S核心蛋白酶体的主要策略是从26S蛋白酶体中去除19S调节亚基。该协议包括两个主要部分:1)的来自表达表位标记的PAG1稳定的转基因品系20S蛋白酶体亲和纯化,的20S蛋白酶(程序AD)的基本组成部分; 2 )体外20S蛋白酶体降解测定法(方法E)。我们预计该协议将提供一种简单有效的方法来研究体外20S蛋白酶体降解,并促进植物中蛋白质代谢的研究。

[背景]蛋白质的降解通常是通过真核生物中的蛋白酶体来实现的。整合的26S蛋白酶体由两个亚颗粒组成:一个或两个末端的19S调节颗粒(RP),用作蛋白酶体激活剂;和20S核心蛋白酶体(CP),执行降解过程。大多数真核蛋白被多聚泛素化并导入26S蛋白酶体进行降解。然而,含有固有蛋白质无序已发现的区域直接通过破坏一个由20S蛋白酶的泛素依赖性降解(本日产等人,2014) ...

Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale
Author:
Date:
2020-04-05
[Abstract]  Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. ... [摘要]  [摘要 ] 人体肠道菌群中的玫瑰菌属和真细菌属在维持人类健康中起着重要作用,部分原因是产生丁酸盐,这是我们结肠上皮细胞的主要能源。但是,由于缺乏基因操作技术,我们对这些细菌的生物化学和生理学的认识受到限制。先前引入玫瑰花属物种的共轭转座子不容易被修饰,极大地限制了它们作为基因修饰平台的适用性。MOD ular质粒穿梭载体先前已经开发了用于梭菌物种,其与共享一个分类次序ř oseburia 和真杆菌,提高这些矢量可以在这些生物体中使用的可能性。在这里,我们描述了一种优化缀合协议使得能够自主复制的质粒的从转印大肠杆菌供体菌株为罗斯氏inulinivorans 和真杆菌rectale 。质粒的模块性质及其通过自主复制在受体细菌中得以维持的能力使其成为研究异源基因表达的理想之选,并成为其他遗传工具(包括反义RNA沉默或II 型移动子中断子基因破坏策略)的平台。

[背景 ] 玫瑰菌和真细菌属人类肠道菌群中含量最高的细菌(Zhernakova 等,2016),它们通过利用饮食和宿主衍生的多糖影响人类健康(Scott 等,2006和2011; Cockburn 等) 。,2015 ; 谢里登等人,2016 )并产生促进健康的代谢物丁酸作为发酵终产物(邓肯等人,2002和2006) 。另外,这些物种能够通过鞭毛调节宿主免疫(Neville ...

Histone Deubiquitination Assay in Nicotiana benthamiana
Author:
Date:
2018-03-05
[Abstract]  Histone modifications are a group of post-translational modifications on histones which can alter chromatin structure and affect gene expression. Histone ubiquitination is a histone modification found in particular on histone H2A and H2B. Histone ubiquitination can be reversed by ubiquitin-specific proteases (UBP). Here, we describe an in vivo assay for histone deubiquitination activity. After infiltrating UBP12 into Nicotiana benthamiana leaves, H2Aub was visualized by immunocytochemistry. Nicotiana benthamiana leaves, which show high agro infiltration efficiency, were used for transient UBP12 expression for a labor- and time-saving protocol. Reduced H2Aub levels indicated histone deubiquitination activity of UBP12. The clear visualization of nuclei of N. ... [摘要]  组蛋白修饰是一组组蛋白翻译后修饰,可以改变染色质结构并影响基因表达。组蛋白泛素化是组蛋白H2A和H2B特异性发现的组蛋白修饰。泛素特异性蛋白酶(UBP)可以逆转组蛋白泛素化。在这里,我们描述了组蛋白去泛素化活性的体内试验。在将UBP12渗入烟草叶片中后,通过免疫细胞化学观察H2Aub。表现出高的农业渗透效率的本氏烟草叶用于瞬时UBP12表达,用于节省劳力和时间的方案。 H2Aub水平降低表明UBP12的组蛋白去泛素化活性。 N的核的清晰可视化。本生叶使得该方法能够通过使用特异性抗体容易地测量体内组蛋白修饰的水平,从而提供强大的蛋白质功能线索。因此,该协议是组蛋白去泛素化活性的体外试验的有力补充。

【背景】组蛋白修饰在调节染色质结构和基因表达中发挥重要作用。 研究最深入的组蛋白修饰包括甲基化,乙酰化,磷酸化,泛素化和sumoylation。 然而,引入或去除特定组蛋白修饰的酶并不总是已知的。 强大的体外试验可以确定组蛋白修饰酶的催化潜能,但是体内试验方法对于确认体外试验的特异性反映抗体的特异性是必要的。 体内活动。 在这里,我们描述了一个灵活的协议来测试植物组织中组蛋白修饰酶的活性。本塞姆氏。 尽管我们使用该方案来测试遍在蛋白特异性蛋白酶(UBP)对泛素化H2A的活性,但它也可以容易地用于其他特异性抗体可用的组蛋白修饰。

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