Separating Inner and Outer Membranes of Escherichia coli by EDTA-free Sucrose Gradient Centrifugation.
Separating Inner and Outer Membranes of Escherichia coli by EDTA-free Sucrose Gradient Centrifugation.
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DOI：
10.21769/bioprotoc.4638
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发表时间：
2023-03-20
期刊：
影响因子：
0.8
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中科院分区：
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革兰氏阴性菌的包膜由外膜 (OM)、肽聚糖细胞壁和内膜 (IM) 组成。 OM和IM具有不同的蛋白质和脂质成分。分离 IM 和 OM 是进一步研究不同位置的脂质和膜蛋白的基本生化程序。溶菌酶/EDTA处理的全膜的蔗糖梯度超速离心是分离革兰氏阴性菌的IM和OM的最广泛使用的方法。然而，EDTA 通常对蛋白质结构和功能有害。在这里，我们描述了一种相对简单的蔗糖梯度超速离心方法来分离大肠杆菌的 IM 和 OM。在该方法中，通过高压微流化床破碎细胞，并通过超速离心收集总细胞膜。然后在蔗糖梯度上分离 IM 和 OM。由于不使用EDTA，该方法有利于后续的膜蛋白纯化和功能研究。
The envelope of Gram-negative bacteria consists of an outer membrane (OM), a peptidoglycan cell wall, and an inner membrane (IM). The OM and IM have different components of proteins and lipids. Separating the IM and OM is a basic biochemical procedure to further study lipids and membrane proteins in different locations. Sucrose gradient ultracentrifugation of lysozyme/EDTA-treated total membrane is the most widely used method to separate the IM and OM of Gram-negative bacteria. However, EDTA is often harmful to protein structure and function. Here, we describe a relatively simple sucrose gradient ultracentrifugation method to separate the IM and OM of Escherichia coli. In this method, the cells are broken by a high-pressure microfluidizer, and the total cell membrane is collected by ultracentrifugation. The IM and OM are then separated on a sucrose gradient. Because EDTA is not used, this method is beneficial for subsequent membrane protein purification and functional study.