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MES hydrate

MES水合物

Company: Sigma-Aldrich
Catalog#: M8250
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Trimolecular Fluorescence Complementation (TriFC) Assay for Direct Visualization of RNA-Protein Interaction in planta
Author:
Date:
2017-10-20
[Abstract]  RNA-Protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA/protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-Protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-Protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) (Schonberger et al., 2012). Target RNA is tagged with 6xMS2 and MCP and RNA binding ... [摘要]  RNA-蛋白质相互作用在各种真核生物过程中起重要作用。 RNA /蛋白质复合物在植物中亚细胞定位的分子成像对于理解这些相互作用至关重要。然而,到目前为止,尚未开发在活植物中形成RNA-蛋白质相互作用的方法。最近,我们开发了一种三分子荧光互补(TriFC)系统,用于在烟草叶中瞬时表达的RNA-蛋白质相互作用的体内可视化。在这种方法中,我们将传统的双分子荧光互补(BiFC)系统与MS2系统(噬菌体MS2外壳蛋白[MCP]及其结合RNA序列[MS2序列])(Schonberger等人,2012)相结合, 。目标RNA用6xMS2标记,MCP和RNA结合蛋白与YFP片段融合。编码这种融合RNA和蛋白质的DNA构建体用土壤杆菌悬浮液渗入烟草叶中。通过共焦显微镜观察体内的RNA-蛋白质相互作用
【背景】近来,多种类型的长非编码RNA(lncRNA)已经被鉴定并显示出在转录调节和染色质修饰中起重要作用(St Laurent等人,2015)。到目前为止,lncRNA介导的功能的大多数分子机制与RNA-蛋白质相互作用密切相关(St ...

Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity
Author:
Date:
2017-10-20
[Abstract]  Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by spectrofluorometric quantification. These techniques are, however, limited to substrates that are available as radiolabeled isotopes or to when the substrate is fluorescent. This prompted us to develop a transport assay where we could in principle detect transport activity for any organic metabolite regardless of its availability as radiolabeled isotope or fluorescence properties.

In this protocol we describe the use of X. laevis oocytes ...
[摘要]  非洲爪蟾卵母细胞是用于表征异源表达的第二活性转运蛋白的广泛使用的模型系统。历史上,研究人员依靠通过闪烁计数或荧光标记的底物通过分光荧光定量测量放射性标记底物的积累来检测运输活动。然而,这些技术仅限于可用作放射性标记的同位素的底物或当底物是荧光时。这促使我们开发了一种运输测定法,我们可以原则上检测任何有机代谢物的转运活性,无论其作为放射性标记的同位素或荧光性质如何。

在这个协议中,我们描述了X的使用。卵母细胞作为表达次要活性转运蛋白的异源宿主,以及如何进行摄取测定,然后通过液相色谱 - 质谱(LC-MS)检测和定量运输的代谢物。我们已经成功地使用这种方法来鉴定和表征称为硫代葡萄糖苷和氰基葡糖苷的植物防御代谢物的转运蛋白(Jørgensen等人,2017),然而该方法可用于表征任何转运蛋白底物可以通过LC-MS检测。

【背景】来自非洲爪蟾蛙(Xenopus laevis)的卵母细胞是用于异源表达和表征膜蛋白(即,转运蛋白和通道)的完整表达系统。 X。卵母细胞表达少量内源性膜蛋白,具有低的背景转运活性。此外,来自植物的次要活性转运蛋白(Boorer等人,1992; Theodoulou和Miller,1995; Nour-Eldin等人,2006),动物(Sumikawa 1981); ...

Isolation and Quantification of Plant Extracellular Vesicles
Author:
Date:
2017-09-05
[Abstract]  Extracellular vesicles (EVs) play an important role in intercellular communication by transporting proteins and RNA. While plant cells secrete EVs, they have only recently been isolated and questions regarding their biogenesis, release, uptake and function remain unanswered. Here, we present a detailed protocol for isolating EVs from the apoplastic wash of Arabidopsis thaliana leaves. The isolated EVs can be quantified using a fluorometric dye to assess total membrane content. [摘要]  细胞外囊泡(EVs)通过传递蛋白质和RNA在细胞间通讯中发挥重要作用。 虽然植物细胞分泌电动汽车,但是它们最近才被孤立,并且关于它们的生物发生,释放,摄取和功能的问题仍然没有得到回答。 在这里,我们提出了一个详细的方案,用于从拟南芥叶片的脱水洗涤中分离EV。 可以使用荧光染料定量分离的EV,以评估总膜含量。
【背景】细胞外囊泡(EVs)是介导蛋白质,脂质和遗传物质的细胞与细胞转移的膜结合结构。由于哺乳动物EVs转运RNA和调节免疫反应的能力,对哺乳动物EV的兴趣已经增长。哺乳动物EV通常被分离用于从培养细胞的培养基中研究,以及生物流体的增长列表(Colombo等,2014)。植物电动车也被认为在免疫反应中起作用,但比较缺乏(An et al。,2007; Davis et al。,2016)。这在很大程度上归因于没有孤立的方法。
  虽然植物EVs自1967年以来一直被观察到,使用透射电子显微镜,但直到2009年才开发出分离方法(Halperin和Jensen,1967)。 ...

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