Summary
This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to logarithmic phase. Cells are collected, their cell walls enzymatically disintegrated and the resulting spheroplasts are homogenized. Mitochondria are pre-fractionated from this homogenate by differential centrifugation. With the focus on further purification, pre-fractionated mitochondria are subjected to ZE-FFE. In ZE-FFE, mitochondria are transported with the buffer flow through the separation chamber and purified from contaminants by specific deflection through a perpendicularly oriented electric field. The purified mitochondria can be collected by centrifugation and used for further experiments and analysis such as sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), immunoblotting, 2-DE or electron microscopy.
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Acknowledgments
The authors thank Dr. E. E. Rojo for critical reading of the manuscript. We also thank Prof. Dr. A. Borst and D. Büringer for their support with electron microscopy. This project was funded by the German Federal Ministry for Education and Research (BMBF) (FKZ: 031U208E, subproject 3; NGFN2 SMP Proteomics, FKZ: 01GR0449, subproject 9, ‘Human Brain’) and the European Union by EU grant LSHG-CT-2003-50520 (INTERACTION PROTEOME).
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Zischka, H., Kinkl, N., Braun, R.J., Ueffing, M. (2008). Purification of Saccharomyces cerevisiae Mitochondria by Zone Electrophoresis in a Free Flow Device. In: Pflieger, D., Rossier, J. (eds) Organelle Proteomics. Methods in Molecular Biology™, vol 432. Humana Press. https://doi.org/10.1007/978-1-59745-028-7_4
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DOI: https://doi.org/10.1007/978-1-59745-028-7_4
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