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Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells

Nature Protocols volume 4, pages 1582–1590 (2009)Cite this article

Abstract

Many cellular processes require the proper cooperation between mitochondria and the endoplasmic reticulum (ER). Several recent works show that their functional interactions rely on dynamic structural contacts between both organelles. Such contacts, called mitochondria-associated membranes (MAMs), are crucial for the synthesis and intracellular transport of phospholipids, as well as for intracellular Ca2+ signaling and for the determination of mitochondrial structure. Although several techniques are available to isolate mitochondria, only few are specifically tuned to the isolation of MAM, containing unique regions of ER membranes attached to the outer mitochondrial membrane and mitochondria without contamination from other organelles (i.e., pure mitochondria). Here we provide optimized protocols to isolate these fractions from tissues and cells. These procedures require 4–5 h and can be easily modified and adapted to different tissues and cell types.

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Figure 1
Figure 2
Figure 3
Figure 4: Pictures of different kinds of pellet and fractions obtained during the isolation process.
Figure 5: Intracellular distributions of p66Shc.

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Acknowledgements

Antibody against CNX was a kind gift from Thomas Simmen. This research was supported by the Polish State Committee for Scientific Research (grant N301 092 32/3407), by the Polish Mitochondrial Network for M.R.W., M.L. and J.D. and by AIRC, UMDF, PRRIITT, FISM and Telethon (GGP09128) grants and local funds from the University of Ferrara to P.P.

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Author notes

  1. Mariusz R Wieckowski and Carlotta Giorgi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland

    Mariusz R Wieckowski, Magdalena Lebiedzinska & Jerzy Duszynski

  2. Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation (ICSI) and Emilia Romagna Laboratory BioPharmaNet, University of Ferrara, Ferrara, Italy

    Carlotta Giorgi & Paolo Pinton

  3. Vita-Salute San Raffaele University, Center of Excellence in Cell Development, and IIT Network, Research Unit of Molecular Neuroscience, Milan, Italy

    Carlotta Giorgi

Authors
  1. Mariusz R Wieckowski
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  2. Carlotta Giorgi
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  3. Magdalena Lebiedzinska
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  4. Jerzy Duszynski
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Contributions

All authors contributed extensively to the work presented in this paper. M.R.W., C.G. and M.L. carried out experiments; M.R.W., J.D. and P.P. analyzed data; M.R.W., C.G. and P.P. wrote the paper.

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Correspondence to Mariusz R Wieckowski or Paolo Pinton.

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Wieckowski, M., Giorgi, C., Lebiedzinska, M. et al. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat Protoc 4, 1582–1590 (2009). https://doi.org/10.1038/nprot.2009.151

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