Collins Carina A, Leslie Michelle E, Peck Scott C, Heese Antje
Division of Biochemistry, Interdisciplinary Plant Group (IPG), University of Missouri, Columbia, MO, 65211, USA.
Methods Mol Biol. 2017;1564:155-168. doi: 10.1007/978-1-4939-6813-8_13.
The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.
质膜（PM）在植物细胞与其环境之间形成一道屏障。处于这个亚细胞位置的蛋白质发挥着多样而复杂的作用，包括感知细胞外信号以协调细胞变化。然而，质膜蛋白的分析常常受到这些蛋白质在总细胞蛋白库中相对丰度较低的限制。传统上用于富集质膜蛋白的技术耗时、繁琐，且需要大量优化。在这里，我们提供了一种简单且可重复的方法，用于从拟南芥幼苗中富集质膜蛋白，起始材料是通过差速离心分离得到的总微粒体膜。为了富集质膜蛋白，用非离子去污剂Brij-58处理总微粒体，以降低污染细胞器蛋白的丰度。该方案与拟南芥中可用的遗传资源相结合，提供了一个强大的工具，将增强我们对质膜蛋白的理解。
