Department of Biochemistry, Cambridge University, Cambridge, UK.
Methods Mol Biol. 2024;2772:115-127. doi: 10.1007/978-1-0716-3710-4_8.
Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis, ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused. A large portion of these vesicles are of sufficiently different surface charge that separation from the majority of Golgi and other contaminants is possible. Here we adapt an earlier ZE-FFE Golgi isolation protocol for the isolation of highly pure ER vesicles and for tracking the migration of peripheral ER vesicles. Isolating ER vesicles of homogeneous surface charge allows multi-omic analyses to be performed on the ER. This facilitates investigations into structure-function relationships within the ER.
自由流动电泳（FFE）是一种分离蛋白质、肽、细胞器和细胞的技术。通过区带电泳（ZE-FFE），根据表面电荷分离细胞器。内质网是拟南芥中唯一尚未使用密度离心、免疫分离或以前应用于植物膜纯化的任何其他方法分离的主要细胞区室。通过使用连续流动电泳，可以聚焦具有相似表面电荷的内质网小泡，这些小泡可能在细胞裂解过程中发生了片段化。这些小泡的很大一部分具有足够不同的表面电荷，因此可以与高尔基体和其他污染物分离。在这里，我们对早期的 ZE-FFE 高尔基分离方案进行了改编，用于分离高度纯净的内质网小泡，并用于跟踪外周内质网小泡的迁移。分离具有均匀表面电荷的内质网小泡可以对内质网进行多组学分析。这有助于研究内质网中的结构-功能关系。
