Summary
This protocol describes the purification of mitochondria from rat liver with the aid of zone electrophoresis in a free flow device (ZE-FFE). Starting from liver homogenate, cell debris and nuclei are removed by low speed centrifugation. A crude mitochondrial fraction is obtained by medium speed centrifugation and is further purified by washing followed by a Nycodenz® gradient centrifugation. Lysosomes and microsomes are located at the upper parts of the gradient, whereas mitochondria are found in the medium part of the gradient. A subsequent purification step with ZE-FFE efficiently removes remaining lysosomes and microsomes and, importantly, damaged mitochondrial structures. The resulting purified mitochondria can be concentrated by centrifugation and used for further experiments. Finally, possible modifications of this protocol with respect to the isolation of pure lysosomes are discussed.
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References
Hannig, K. and Heidrich, H. g. (1974) The use of continuous preparative free-flow electrophoresis for dissociating cell fractions and isolation of membranous components. Meth. Enzymol. 31, 746–61.
Heidrich, H. g. , Stahn, R., and Hannig, K. (1970) The surface charge of rat liver mitochondria and their membranes. Clarification of some controversies concerning mitochondrial structure. J. Cell Biol. 46(1), 137–50.
Stahn, R., Maier, K. P., and Hannig, K. (1970) A new method for the pretion of rat liver lysosomes. Setion of cell organelles of rat liver by carrier-free continuous electrophoresis. J. Cell Biol. 46(3), 576–91.
Hannig, K. and Wrba, H. (1964) Isolation of Vital Tumor Cells by Carrier-Free Electrophoresis. Z. Naturforsch. B. 19, 860.
Murayama K., Fujimura T., Morita, M., and Shindo, N. (2001) One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles. Electrophoresis 22(14), 2872–80.
Klein, D., Lichtmannegger, J., Heinzmann, U., and Summer, K. H. (1998) Association of copper to metallothionein in hepatic lysosomes of Long-Evans cinnamon (LEC) rats during the development of hepatitis. Eur. J. Clin. Invest. 28(4), 302–10.
Klein, D., Lichtmannegger, J., Heinzmann, U., Muller-Hocker, J., Michaelsen, S., and Summer, K. H. (2000) Dissolution of copper-rich granules in hepatic lysosomes by D-penicillamine prevents the development of fulminant hepatitis in Long-Evans cinnamon rats. J. Hepatol. 32(2), 193–201.
Fuller, K.M. and Arriaga, E.A. (2004) Capillary electrophoresis monitors changes in the electrophoretic behavior of mitochondrial pretions. J. Chromatogr. B 806(2), 151–9.
Zischka, H., Weber, g. , Weber, P. J., Posch, A., Braun, R. J., Buhringer, D., Schneider, U., Nissum, M., Meitinger, T., Ueffing, M., and Eckerskorn, C. (2003) Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis. Proteomics 3(6), 906–16.
Weber, g. and Bocek, P. (1996) Optimized continuous flow electrophoresis. Electrophoresis 17(12), 1906–10.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259), 680–5.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U S A 76(9), 4350–4.
Lewis, P. R. and Shute, C.C. (1969) An electron-microscopic study of cholinesterase distribution in the rat adrenal medulla. J. Microsc. 89(2), 181–93.
Ericson, I. (1974) Determination of the isoelectric point of rat liver mitochondria by cross-partition. Biochim. Biophys. Acta 356(1), 100–7.
Acknowledgements
The authors would like to thank Drs. E. E. Rojo and g. Greif for critical reading of the manuscript. HZ would especially like to thank Dr. M. Ueffing for his support.
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Zischka, H. et al. (2008). Isolation of Highly Pure Rat Liver Mitochondria with the Aid of Zone-Electrophoresis in a Free Flow Device (ZE-FFE). In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 424. Humana Press. https://doi.org/10.1007/978-1-60327-064-9_26
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DOI: https://doi.org/10.1007/978-1-60327-064-9_26
Publisher Name: Humana Press
Print ISBN: 978-1-58829-722-8
Online ISBN: 978-1-60327-064-9
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