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Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue
Published on: March 23, 2015
BAK/BAX activation and cytochrome c release assays using isolated mitochondria
Thibaud T Renault1, Konstantinos V Floros, Jerry E Chipuk
1Icahn School of Medicine at Mount Sinai, Department of Oncological Sciences, One Gustave L. Levy Place, Box 1130, New York, NY 10029, USA.
Methods (San Diego, Calif.)
|April 10, 2013
Summary
This study details methods for analyzing mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release, crucial steps in apoptosis. It focuses on the roles of BAK and BAX proteins in triggering these events using isolated mitochondria.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is essential for development and tissue homeostasis.
- The mitochondrial pathway of apoptosis is initiated by the permeabilization of the outer mitochondrial membrane (OMM).
- Pro-apoptotic BCL-2 family proteins, BAK and BAX, are key regulators that form pores in the OMM, leading to mitochondrial outer membrane permeabilization (MOMP).
Purpose of the Study:
- To describe and validate in vitro techniques for studying MOMP and cytochrome c release.
- To elucidate the functional requirements for BAK and BAX activation in isolated mitochondria.
- To biochemically define the interplay between BCL-2 family proteins and mitochondrial physiology.
Main Methods:
- Utilizing freshly isolated mitochondria from various sources.
- Performing in vitro assays to induce and measure MOMP.
- Conducting cytochrome c release assays to quantify apoptotic signaling.
Main Results:
- Established methodologies to assess MOMP and cytochrome c release in isolated mitochondrial systems.
- Provided techniques to implicate BAK and BAX activity in these apoptotic processes.
- Demonstrated the utility of isolated mitochondria for studying BCL-2 family function.
Conclusions:
- Isolated mitochondria are a powerful tool for dissecting the molecular mechanisms of apoptosis.
- The described methods enable detailed investigation of BAK and BAX activation and their role in MOMP.
- This work facilitates a deeper understanding of mitochondrial regulation of apoptosis.
