Journal
NATURE PROTOCOLS
Volume 3, Issue 6, Pages 965-976Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2008.61
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Funding
- Austrian Cancer Society/Tyrol [KU-911/15-1]
- Deutsche Forschungsgemeinschaft [SCHR-562/4-3]
- BMBF [01GZ0704]
- Agence National de la Recherche [BLAN07-2_188128]
- Estonian Science Foundation [Ndegrees 6142, 7117]
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Analysis of mitochondrial function is central to the study of intracellular energy metabolism, mechanisms of cell death and pathophysiology of a variety of human diseases, including myopathies, neurodegenerative diseases and cancer. However, important properties of mitochondria differ in vivo and in vitro. Here, we describe a protocol for the analysis of functional mitochondria in situ, without the isolation of organelles, in selectively permeabilized cells or muscle fibers using digitonin or saponin. A specially designed substrate/inhibitor titration approach allows the step-by-step analysis of several mitochondrial complexes. This protocol allows the detailed characterization of functional mitochondria in their normal intracellular position and assembly, preserving essential interactions with other organelles. As only a small amount of tissue is required for analysis, the protocol can be used in diagnostic settings in clinical studies. The permeabilization procedure and specific titration analysis can be completed in 2 h.
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