| RAB21 Activity Assay Using GST-fused APPL1
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Author:
Date:
2016-02-20
[Abstract] The Rab family of small GTPases are essential regulators of membrane trafficking events. As with other small GTPase families, Rab GTPases cycle between an inactive GDP- bound state and an active GTP-bound state. Guanine nucleotide exchange factors (GEFs) promote Rab activation with the exchange of bound GDP for GTP, while GTPase-activating proteins (GAPs) regulate Rab inactivation with GTP hydrolysis. Numerous methods have been established to monitor the activation status of Rab GTPases. Of those, FRET-based methods are used to identify when and where a Rab GTPase is activated in cells. Unfortunately, the generation of such probes is complex, and only a limited number of Rabs have been probed this way. Biochemical purification of activated Rabs from cell or tissue extracts is easily ...
[摘要] 小GTP酶的Rab家族是膜运输事件的必要调节剂。与其他小GTP酶家族一样，Rab GTP酶在不活动的GDP结合状态和活性GTP结合状态之间循环。鸟嘌呤核苷酸交换因子（GEF）促进Rab激活与交换绑定GDP GTP，而GTPase激活蛋白（GAP）调节Rab失活与GTP水解。已经建立了许多方法来监测Rab GTPases的活化状态。其中，基于FRET的方法用于鉴定细胞中Rab GTPase在何时和何地被激活。不幸的是，这种探针的产生是复杂的，并且只有有限数量的Rab已经以这种方式探测。来自细胞或组织提取物的活化的Rabs的生物化学纯化通过使用已知的Rab效应结构域以下拉特定的GTP结合的Rab形式是容易实现的。虽然这种方法不是理想的详细的亚细胞定位，它可以提供Rab活动的时间分辨率。越来越多的特异性效应物的鉴定现在允许在特定条件下测试许多Rab GTP酶的活化水平。在这里，我们描述了一种亲和纯化方法使用GST融合APPL1（一种已知的RAB21效应）来测试哺乳动物细胞中的RAB21激活。该方法成功地用于测定RAB21激活状态下营养丰富与饥饿条件下的变化，并测试在此过程中MTMR13 RAB21 GEF的需求。
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| VAMP8-3xHA Uptake Assay in HeLa Cells
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Author:
Date:
2016-02-20
[Abstract] Transmembrane proteins are rarely exclusively localized to a specific vesicle or an organelle. Most transmembrane proteins undergo complicated trafficking routes. Thus, transmembrane proteins are under constant flux, and at steady state, found on a variety of vesicles or organelles. This characteristic makes the study of their trafficking routes complex, since at any given moment, different molecules are often being trafficked in opposing directions. Pulse-chase experiments can temporally track a specific pool of a transmembrane protein of interest, allowing for the kinetic description of its trafficking route. This type of technique has been used extensively to follow a large array of plasma membrane localized proteins (Diril et al., 2006; Jean et al., 2010). Here, we ...
[摘要] 跨膜蛋白很少专门定位于特定的囊泡或细胞器。大多数跨膜蛋白经历复杂的运输路线。因此，跨膜蛋白在恒定通量下，并在稳定状态，发现在各种囊泡或细胞器。这个特征使得他们的贩运路线的研究复杂，因为在任何给定时刻，不同的分子通常在相反的方向上被贩运。脉冲追踪实验可以暂时跟踪感兴趣的跨膜蛋白的特定池，允许其运输路线的动力学描述。这种类型的技术已广泛用于跟踪大量的质膜定位蛋白质（Diril等人，2006; Jean等人，2010）。在这里，我们描述了一种方法，允许研究VAMP8贩运从质膜到内溶酶体隔室。该方法用于描述 MTMR13 和 RAB21 在调节VAMP8向内溶酶体的运输中的作用（Jean等人，，2015）。
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| Preparation of Candida albicans Biofilms for Transmission Electron Microscopy
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Author:
Date:
2013-07-20
[Abstract] Transmission Electron Microscopy is a form of microscopy that allows for imaging of distinct portions of an individual cell. For Candida albicans biofilms, it is often used to visualize the cell walls of fixed samples of yeast and hyphae. This protocol describes how to grow, harvest, and fix Candida albicans biofilms in preparation for Transmission Electron Microscopy.
[摘要] 透射电子显微术是允许对单个细胞的不同部分成像的显微镜检查的形式。 对于白色念珠菌生物膜，它通常用于可视化酵母和菌丝的固定样品的细胞壁。 此协议描述如何生长，收获和修复白色念珠菌生物膜准备透射电子显微镜。
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