Immunopurification of Golgi vesicles by magnetic sorting
Immunopurification of Golgi vesicles by magnetic sorting
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DOI：
10.1016/s0022-1759(01)00546-4
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发表时间：
2002-02-01
影响因子：
2.2
通讯作者：
Wolff, D
中科院分区：
文献类型：
--
作者：
Mura, CV;Becker, ML;Wolff, D
我们设计了一种方法，允许分离高度纯化的高尔基体囊泡剥夺内质网（ER），主要污染物的高尔基体组分。为此，我们制备了一种兔多克隆抗体对酶乙酰肝素氨基葡萄糖N-脱乙酰基酶/N-磺基转移酶（HSST），高尔基体的特异性标志物的细胞溶质N-末端寡肽。间接免疫荧光显微镜证实了HSST的高尔基体定位。免疫电镜显示抗体与高尔基体囊泡结合，并通过磁性分选进行免疫纯化。通过磁分选纯化的高尔基体囊泡显示存在HSST和p28，p28是顺式高尔基体上的一种完整膜蛋白，也用作特异性高尔基体标记。纯化的材料缺乏钙网蛋白，一种特异性ER标记物。这种纯化方法将允许改进需要高度纯化的高尔基体膜的研究，例如特异性受体的鉴定和高尔基体膜离子通道的电生理学表征，这些研究到目前为止已被ER膜污染所危害。(C)2002年爱思唯尔科学有限公司保留所有权利。
We have designed a method that permits to isolate highly purified Golgi vesicles deprived of endoplasmic reticulum (ER), main contaminant of Golgi fractions. To this end, we prepared a rabbit polyclonal antibody against the cytosolic N-terminal oligopeptide of the enzyme heparan glucosaminyl N-deacetylase/N-sulphotransferase (HSST), a specific marker for Golgi apparatus. The Golgi localization of HSST was confirmed by indirect immunofluorescence microscopy. The antibody binding to Golgi vesicles was demonstrated by immunoelectronmicroscopy and allowed the immunopurification by magnetic sorting. Golgi vesicles subjected to purification by magnetic sorting showed the presence of HSST and p28, which is an integral membrane protein on the cis-Golgi also used as a specific Golgi marker. The purified material was devoid of calreticulin, a specific ER marker. This purification method will allow to improve studies requiring highly purified Golgi membranes such as identification of specific receptors and the electrophysiological characterization of Golgi membrane ion channels, which have been jeopardized up to now by ER membrane contamination. (C) 2002 Elsevier Science B.V. All fights reserved.