Investigating bax subcellular localization and membrane integration
- Author(s)
- Dewson, G;
- Details
- Publication Year 2015-05-01,Volume 2015,Issue #5,Page 467-471
- Journal Title
- Cold Spring Harb Protoc
- Publication Type
- Journal Article
- Abstract
- 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.
- Publisher
- CSHLP
- Research Division(s)
- Cell Signalling And Cell Death
- PubMed ID
- 25934936
- Publisher's Version
- https://doi.org/10.1101/pdb.prot086447
- NHMRC Grants
- NHMRC/637335,
- ARC Grants
- ARC/FT100100791,
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2015-05-22 11:19:35
Last Modified: 2016-01-21 08:49:19