| Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells
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Author:
Date:
2021-03-05
[Abstract] Monitoring vesicle trafficking is an excellent tool for the evaluation of protein dynamics in living cells. Such study is key for the understanding of protein sorting and secretion. Recent developments in microscopy, as well as new methodologies developed to study synchronized trafficking of proteins, allowed a better understanding of signaling, regulation and trafficking dynamics at the secretory pathway. One of the most helpful tools so far developed is the Retention Using Selective Hooks (RUSH) system, a methodology that facilitates the evaluation of synchronized cargo trafficking by monitoring fluorescent vesicles in cells upon biotin addition. Here we present a protocol that allows the quantitative evaluation of protein cargo trafficking at different fixed time points and an analytic ...
[摘要] [摘要]监测囊泡运输是评估活细胞中蛋白质动力学的出色工具。这项研究对于理解蛋白质的分类和分泌至关重要。显微镜的最新发展，以及研究同步蛋白运输的新方法，使人们对蛋白质的理解有了更深入的了解。 分泌途径中的信号传导，调节和运输动态。迄今为止，开发的最有用的工具之一是使用选择性钩子保留（RUSH）系统，该方法可通过在添加生物素后监测细胞中的荧光囊泡来促进同步货物运输的评估。在这里，我们提出了一个协议，允许蛋白质贩卖货物在不同的固定时间点的定量评价和ANALY抽动的方法，使一个更好的检查特定货物走私力度的分泌途径。
图形摘要：
哺乳动物细胞中RUSH分选测定的示意图
[背景技术[ 0002 ]监测囊泡运输是评估活细胞中分泌蛋白动力学的极好的工具。鉴于哺乳动物新合成蛋白质总量中约有30％遵循分泌途径（Pfeffer，2010; Boncompain and Weigel，2018），因此研究其运输动力学是了解蛋白质分类和分泌的关键。遵循分泌途径的蛋白质经历了多个成熟步骤，从内质网开始，沿着不同的高尔基体堆栈运输，直到到达反式高尔基体网络（TGN），即分选站。在TGN中，它们最终被包装到囊泡中，并开始到达最终目的地的路线（Glick和Luini，2011； Pantazopoulou和Glick，2019）。
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| Seed Sterilization and Seedling Growth on Plates in the Model Grass Brachypodium distachyon
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Author:
Date:
2020-08-05
[Abstract] Brachypodium distachyon is a model grass closely related to wheat and barley. New resources and methods are being developed and Brachypodium is becoming a popular model for developmental biology, cell wall biology, (phylo) genomics, and genome evolution (Scholthof et al., 2018). Sterilization of seeds and seedling growth on plates is the first step to start working with Brachypodium. This is a cheap and straightforward protocol that was successfully used before (Raissig et al., 2016 and 2017).
[摘要] [摘要] 远臂足是一种与小麦和大麦关系密切的模式草。新的资源和方法正在开发中，短尾水母正在成为发育生物学、细胞壁生物学、基因组学和基因组进化的一种流行模式（Scholthof等人，2018年）. 种子消毒和幼苗在培养皿上生长是开始使用短足类的第一步。这是一个廉价而直接的协议，以前曾成功使用过（Raissig等人，2016年和2017年）.
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| Use of SCRI Renaissance 2200 (SR2200) as a Versatile Dye for Imaging of Developing Embryos, Whole Ovules, Pollen Tubes and Roots
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Author:
Date:
2016-09-20
[Abstract] Confocal laser scanning microscopy in combination with fluorescent proteins is a powerful tool for the study of sexual reproduction and other developmental processes in plants. In order to understand the origin and localization of fluorescent signals in a complex tissue, staining of cell outlines is often mandatory. Cell wall staining with SCRI Renaissance 2200 (SR2200) has recently been described as a method of choice to study plant reproductive processes (Musielak et al., 2015). In this protocol, we present detailed instructions on the use of SR2200 to stain cell walls in different Arabidopsis tissues.
[摘要] 共聚焦激光扫描显微镜结合荧光蛋白是研究植物中有性繁殖和其他发展过程的有力工具。 为了理解复杂组织中荧光信号的起源和定位，细胞轮廓的染色通常是强制性的。 最近已经描述了使用SCRI Renaissance 2200（SR2200）的细胞壁染色作为研究植物繁殖过程的选择方法（Musielak等人，2015）。 在该协议中，我们提供关于使用SR2200在不同的拟南芥组织中染色细胞壁的详细说明。
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