Schaller G Eric
Department of Biological Sciences, Life Sciences Center, Dartmouth College, 78 College Street, Hanover, NH, 03755, USA.
Methods Mol Biol. 2017;1573:113-131. doi: 10.1007/978-1-4939-6854-1_10.
Ethylene receptors and other elements of the ethylene-signal transduction pathway localize to membranes of the endoplasmic reticulum (ER). New players in the ethylene signaling pathway continue to be discovered and so it is important to have methods by which to diagnose their cellular localization. Two methods for microsome isolation and fractionation are described here that can assist in determining if a protein localizes to the ER: aqueous two-phase partitioning and equilibrium density-gradient centrifugation. Two-phase partitioning serves to purify plasma membrane away from other cellular membranes and can thus discriminate whether a protein is localized to the plasma membrane or not. Equilibrium density-gradient centrifugation is particularly useful for resolving if a protein is localized to the ER. Ribosomes are associated with the rough ER in the presence of Mg but are stripped away when Mgis removed from the medium, resulting in a reduction in the ER membrane density and a diagnostic shift in migration when analyzed by equilibrium density-gradient centrifugation. A method for growing plants in liquid culture is also provided because these microsomal membranes exhibit consistent fractionation by both two-phase partitioning and density-gradient centrifugation.
乙烯受体和乙烯信号转导途径的其他元件定位于内质网（ER）膜。乙烯信号通路中的新成员不断被发现，因此拥有诊断其细胞定位的方法很重要。本文描述了两种用于微粒体分离和分级的方法，可帮助确定蛋白质是否定位于内质网：水相两相分配法和平衡密度梯度离心法。两相分配法用于将质膜与其他细胞膜分离，从而可以区分蛋白质是否定位于质膜。平衡密度梯度离心法对于确定蛋白质是否定位于内质网特别有用。在有镁存在的情况下，核糖体与糙面内质网相关联，但当从培养基中去除镁时，核糖体会被剥离，导致内质网膜密度降低，在通过平衡密度梯度离心分析时迁移出现诊断性变化。还提供了一种在液体培养中种植植物的方法，因为这些微粒体膜通过两相分配法和密度梯度离心法都表现出一致的分级。
