Isolation and immobilization of various plastid subtypes by magnetic immunoabsorption
Isolation and immobilization of various plastid subtypes by magnetic immunoabsorption
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DOI：
10.1046/j.1365-313x.1994.6050767.x
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发表时间：
1994-11
期刊：
影响因子：
7.2
通讯作者：
A. Kausch;B. Bruce
中科院分区：
文献类型：
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作者：
A. Kausch;B. Bruce
总结 已经制备了抗体，其免疫定位于豌豆叶绿体包膜的外膜并导致分离的叶绿体凝集。该抗血清与来自单子叶和双子叶植物的多种质体形式具有免疫反应性。已经测试了此类抗体是否可以有效地用于从全细胞裂解物中分离和固定质体。已开发出一种系统，用于使用生物素化抗体和链霉亲和素磁性纳米颗粒对各种形式的高等植物质体进行免疫标记，然后在 0.6 特斯拉高梯度磁场中分离质体。使用这种磁性免疫吸附程序，可以从几种植物物种的全细胞裂解物中实现有色体、淀粉体和叶绿体的高度阳性富集。这些质体的完整性已通过细胞器蛋白质合成、14C-ADP-葡萄糖摄取、流式细胞术、体外合成前体输入和质体包膜的 FITC 阳离子化铁蛋白染色进行了检查。蛋白质印迹分析显示玉米胚乳中胞浆蔗糖合酶的淀粉体显着富集。全细胞裂解物中亚细胞结构的磁免疫吸附是一种新方法，可用于对多种生物体的许多不同细胞区室进行体外分析。
Summary Antibodies have been prepared which immuno-localize to the outer membrane of the pea chloroplast envelope and cause agglutination of isolated chloroplasts. This antisera is immunoreactive with a variety of plastid forms from both monocotyledonous and dicotyledonous plants. Whether such antibodies might be effectively used for isolation and immobilization of plastids from whole cell lysates has been tested. A system has been developed for immunolabeling various forms of higher plant plastids with biotinylated antibody and streptavidin magnetic nano-particles followed by separation of the plastids in a 0.6 Tesla high gradient magnetic field. Using this magnetic immunoabsorption procedure it has been possible to achieve a high degree of positive enrichment for chromoplasts, amyloplasts, and chloroplasts from whole cell lysates of several plant species. The integrity of these plastids has been examined by in organellar protein synthesis, 14C-ADP-glucose uptake, flow cytometry, in vitro synthesized precursor import and FITC-cationized ferritin staining of the plastid envelope. Western blot analysis showed significant enrichment for amyloplasts from cytosolic sucrose synthase in maize endosperm. Magnetic immunoabsorption of subcellular structures from whole cell lysates is a new method that may be useful in the in vitro analysis of many different cellular compartments from a wide range of organisms.