Investigating Bax subcellular localization and membrane integration.
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.
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.
Grant
G
Dewson
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002478:Cells, Cultured; D002498:Centrifugation; D003600:Cytosol / Q000737:chemistry*; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D051379:Mice; D051336:Mitochondrial Membranes / Q000737:chemistry*; D051028:bcl-2-Associated X Protein / Q000032:analysis*
D003902:Detergents; D051028:bcl-2-Associated X Protein