Analysis of Arf GTP-binding protein function in cells.
Analysis of Arf GTP-binding protein function in cells.
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DOI：
10.1002/0471143030.cb1412s48
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发表时间：
2010-09
影响因子：
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通讯作者：
Donaldson, Julie G
中科院分区：
文献类型：
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作者：
Cohen, Lee Ann;Donaldson, Julie G
本单元描述了可用于研究细胞中adp -核糖基化因子（Arf） gtp结合蛋白功能的技术和方法。哺乳动物有6种Arfs和更多的Arfs样蛋白（Arls），这些蛋白在从酵母到人类的真核生物中都是保守的。与所有GTP酶一样，Arfs在GTP结合、无活性和GTP结合的活性构象之间循环，由鸟嘌呤核苷酸交换因子（GEFs）和GTP酶激活蛋白（gap）分别催化GTP结合和水解。在这里，我们描述了可以用来检查细胞中Arf和Arl蛋白的定位和功能的方法（方案1）。我们还提供了一种简单的方案，使用下拉试验来测量细胞中特定Arf蛋白的激活（gtp结合）（方案2）。然后，我们讨论了可用于评估细胞中gef和gap功能的方法（方案3）。
This unit describes techniques and approaches that can be used to study the functions of the ADP-ribosylation factor (Arf) GTP-binding proteins in cells. There are 6 mammalian Arfs and many more Arf-like proteins (Arls) and these proteins are conserved in eukaryotes from yeast to man. Like all GTPases, Arfs cycle between GDP-bound, inactive and GTP-bound active conformations, facilitated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that catalyze GTP binding and hydrolysis respectively. Here we describe approaches that can be taken to examine the localization and function of Arf and Arl proteins in cells (Protocol 1). We also provide a simple protocol for measuring activation (GTP-binding) of specific Arf proteins in cells using a pull-down assay (Protocol 2). We then discuss approaches that can be taken to assess function of GEFs and GAPs in cells (Protocol 3).