Abstract
We recently reported a new method to isolate functionally intact mitochondria from cell culture and small tissue samples (Schmitt et al., Anal Biochem 443(1):66–74, 2013). This method comprises a semi-automated cell rupture, termed pump controlled cell rupture system (PCC), which can be precisely adjusted to the specific cellular source of isolation and which can be tightly controlled (Schmitt et al., Anal Biochem 443(1):66–74, 2013). Here we provide a detailed hands-on protocol of this PCC method which results in an efficient cell breakage but preserving the mitochondrial integrity. Upon subsequent purification steps, the obtained mitochondrial fraction meets the quality and purity required for molecular analyses, e.g. proteomic comparisons, as well as for biochemical analyses, e.g. determination of diverse enzymatic activities.
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Acknowledgement
We would like to acknowledge E.E. Rojo and A. Simmons for critical reading of the manuscript. This study was supported in parts by the Deutsche Forschungsgemeinschaft (DFG) grant RU742/6-1 to H.Z.
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Schmitt, S., Eberhagen, C., Weber, S., Aichler, M., Zischka, H. (2015). Isolation of Mitochondria from Cultured Cells and Liver Tissue Biopsies for Molecular and Biochemical Analyses. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_8
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DOI: https://doi.org/10.1007/978-1-4939-2550-6_8
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2549-0
Online ISBN: 978-1-4939-2550-6
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