Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences.
Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences;
J Vis Exp. 2021 Aug 12(174). doi: 10.3791/62119.
This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.
本方案描述了一种使用去污剂、机械裂解和等密度梯度离心相结合的方法从哺乳动物细胞中获得亚细胞蛋白组分的方法。该方法的主要优点是它不依赖于单一去污剂溶解的方法来获得亚细胞组分。这使得有可能将质膜与细胞的其他膜结合细胞器分离。这种方法将通过一种可重复、可扩展和选择性的方法促进细胞中蛋白质定位的确定。该方法已成功用于从人单核细胞系 U937 中分离细胞质、核、线粒体和质膜蛋白。尽管该方案已针对该细胞系进行了优化，但它可能是其他细胞系亚细胞分级分离的合适起点。讨论了该方案的潜在缺陷以及如何避免这些缺陷，以及可能需要考虑用于其他细胞系的改变。
