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Evolve® 512 EMCCD Camera

公司名称: Photometrics
产品编号: Evolve® 512
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Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay
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2017-11-20
[Abstract]  Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP. Given that GalT is recycled to the cisterna where it is located at steady state, GalT-containing vesicles target GalT-containing cisternal membranes. Golgi membranes were therefore isolated from GalT-CFP expressing cells, ... [摘要]  高尔基体内的逆行囊泡运输被用来运送和分类高尔基酶到适当的池内位置。建立了一个检测方法来研究无细胞系统中囊泡靶向的分子细节。生成了稳定的细胞系,其中反式 - 高尔基酶半乳糖基转移酶(GalT)用CFP或YFP标记。考虑到GalT被循环到稳定状态的小池中,含有GalT的囊泡将靶向含有GalT的池内膜。因此从表达GalT-CFP的细胞分离高尔基体膜,而从GalT-YFP表达细胞制备囊泡。在胞质溶胶和能量再生混合物的存在下,在37℃孵育CFP标记的高尔基体和YFP标记的囊泡,与在冰上孵育相比,在混合物的荧光成像后CFP-YFP共定位显着增加。该测定被验证需要能量,蛋白质和生理学重要的运输组分,如Rab GTP酶和保守寡聚体高尔基体系复合物。该测定法可用于调查影响高尔基体运输机器的生理和病理变化,特别是囊泡束缚。

【背景】细胞内囊泡靶向的分子机制对于解释糖基化稳态,神经递质释放,信号受体的调节和营养摄取等方面的解释是重要的(Ungar和Hughson,2003; ...

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