McCaig William D, Deragon Matthew A, Haluska Robert J, Hodges Alexa L, Patel Payal S, LaRocca Timothy J
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. 2019 Jan 18(143). doi: 10.3791/59022.
In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.
在本实验方案中，我们详细介绍了一种无需使用超速离心或非特异性去污剂即可获得U937细胞亚细胞组分的方法。该方法利用低渗缓冲液、洋地黄皂苷、机械裂解和差速离心来分离细胞质、线粒体和质膜。该过程可根据研究人员的需求进行规模调整，成本低廉且操作简单。此方法使研究人员无需使用专业离心机和商业试剂盒就能确定细胞中的蛋白质定位，而这两者可能价格昂贵。我们已成功运用该方法分离人单核细胞系U937中的胞质、质膜和线粒体蛋白。
