Dewson Grant
Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia;Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
Cold Spring Harb Protoc. 2015 May 1;2015(5):467-71. doi: 10.1101/pdb.prot086447.
Bax is a pivotal effector of apoptosis responsible for permeabilization of the mitochondrial outer membrane (MOM). A key event in mitochondrial damage is the translocation of Bax from the cytosol to the MOM. A simple and effective method for assessing the cytosol vs. mitochondrial localization of Bax is digitonin fractionation, which uses a low concentration of detergent to permeabilize the plasma membrane without damaging intracellular membranes. This allows separation of the cytosol (light membranes) from the heavy membranes (with mitochondria and nuclei) by centrifugation. Localization of Bax can then be assessed by immunoblotting. To further differentiate membrane-integrated Bax from that which is peripherally associated, carbonate extraction of the membrane fraction can be performed before immunoblotting. Treatment of membranes at high pH disrupts protein-protein interactions, whereas protein-lipid interactions are largely retained, although membrane integrity is lost.
Bax是细胞凋亡的关键效应因子，负责线粒体外膜（MOM）的通透性改变。线粒体损伤的一个关键事件是Bax从细胞质转移到MOM。一种评估Bax在细胞质与线粒体定位的简单有效方法是洋地黄皂苷分级分离法，该方法使用低浓度去污剂使质膜通透而不损伤细胞内膜。这样通过离心可将细胞质（轻膜组分）与重膜组分（含线粒体和细胞核）分离。然后可通过免疫印迹法评估Bax的定位。为了进一步区分整合到膜上的Bax和外周结合的Bax，可在免疫印迹之前对膜组分进行碳酸盐抽提。在高pH值下处理膜会破坏蛋白质-蛋白质相互作用，而蛋白质-脂质相互作用虽因膜完整性丧失而大部分保留。
