Investigating Bax subcellular localization and membrane integration.
Cold Spring Harb Protoc | Published : 2015
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 tha..View full abstract