Hansen Sara Fasmer, Ebert Berit, Rautengarten Carsten, Heazlewood Joshua L
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.
Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94702, USA.
Methods Mol Biol. 2016;1496:91-109. doi: 10.1007/978-1-4939-6463-5_8.
The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization of proteins by fluorescent tags after their identification by tandem mass spectrometry.
植物高尔基体在分泌途径中起核心作用，是细胞内大分子组装和加工的主要场所。高尔基体的堆叠膜结构及其与细胞骨架和内质网的相互作用，历来使得该细胞器的分离和纯化变得困难。密度离心法通常用于从植物微粒体制备物中富集高尔基体膜，除了一些小的改进外，该方法仍被广泛应用。在这里，我们概述了从拟南芥细胞悬浮培养物中富集高尔基体膜的方法，该方法可用于研究该细胞器的蛋白质组。我们还提供了一个有用的工作流程，用于对多次分析所得的蛋白质组学数据进行检查。最后，我们重点介绍了一种简单的技术，用于在通过串联质谱鉴定蛋白质后，通过荧光标签验证蛋白质的亚细胞定位。
