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Isolation of plasma membrane–associated membranes from rat liver

Nature Protocols volume 9, pages 312–322 (2014)Cite this article

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Abstract

Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane–associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8–10 h, and it can be easily modified and adapted to other tissues and cell types.

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Figure 1: Timeline and schematic steps for crude PM isolation from rat liver (Steps 1–18).
Figure 2
Figure 3: Images of different types of pellets and fractions obtained during the isolation process.
Figure 4: Intracellular distribution of SIGMAR1 and other markers.

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Acknowledgements

This research was supported by grants from the Polish National Science Centre (UMO-2011/01/M/NZ3/02128), Iuventus Plus (UMO-0531/IP1/2011/71) and Polish Ministry of Science and Higher Education grant W100/HFSC/2011 to J.M.S., M.L., A.W., J.D. and M.R.W.; by the Italian Association for Cancer Research to C.G. and P.P.; and by Telethon (GGP11139B), the Italian Ministry of Education, University and Research, and the Italian Ministry of Health to P.P. J.M.S. was also supported by a Ph.D. fellowship from The Foundation for Polish Science (FNP), and by the EU, the European Regional Development Fund and the 'Innovative economy' Operational Programme. M.L. was supported by the 'Mobility Plus' fellowship from the Polish Ministry of Science and Higher Education.

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Authors and Affiliations

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

    Jan M Suski, Magdalena Lebiedzinska, Aleksandra Wojtala, Jerzy Duszynski & Mariusz R Wieckowski

  2. Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Interdisciplinary Center for the Study of Inflammation (ICSI) and Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy

    Jan M Suski, Magdalena Lebiedzinska, Carlotta Giorgi & Paolo Pinton

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  1. Jan M Suski
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Contributions

J.M.S., M.L., A.W. and C.G. carried out all experimental steps and performed characterization of the isolated subcellular fractions. J.M.S., J.D., P.P. and M.R.W. developed the concept, designed experiments and wrote the paper. All authors discussed the results and commented on the manuscript at all stages.

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Correspondence to Mariusz R Wieckowski.

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The authors declare no competing financial interests.

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Suski, J., Lebiedzinska, M., Wojtala, A. et al. Isolation of plasma membrane–associated membranes from rat liver. Nat Protoc 9, 312–322 (2014). https://doi.org/10.1038/nprot.2014.016

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