Isolation of plasma membrane-associated membranes from rat liver
Isolation of plasma membrane-associated membranes from rat liver
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DOI：
10.1038/nprot.2014.016
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发表时间：
2014-02-01
期刊：
影响因子：
14.8
通讯作者：
Wieckowski, Mariusz R.
中科院分区：
文献类型：
--
作者：
Suski, Jan M.;Lebiedzinska, Magdalena;Wieckowski, Mariusz R.
细胞内细胞器之间的动态相互作用需要膜结构的特定功能对接。所涉及的细胞器密切接触，但不融合，从而产生显著的微域；这些微域允许细胞器之间的快速通信。质膜相关膜(PAM)是质膜(PM)与内质网(ER)和线粒体相互作用的微区，是调节蛋白质、脂类、离子和代谢物运输的动态结构。由于它们在许多关键的细胞过程中的作用，这些结构最近引起了人们的极大兴趣。在这里，我们提供了一种从大鼠肝脏中分离PAM、PM和ER组分的优化方案，该方案基于一系列差速离心法，然后在不连续的蔗糖梯度上分离粗PM。该程序需要类似于8-10小时，并且可以很容易地修改和适应其他组织和细胞类型。
Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires similar to 8-10 h, and it can be easily modified and adapted to other tissues and cell types.