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Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation

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Peroxisomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2643))

Abstract

Sophisticated organelle fractionation strategies were the workhorse of early peroxisome research and led to the characterization of the principal functions of the organelle. However, even in the era of molecular biology and “omics” technologies, they are still of importance to unravel peroxisome-specific proteomes, confirm the localization of still uncharacterized proteins, analyze peroxisome metabolism or lipid composition, or study their protein import mechanism. To isolate and analyze peroxisomes for these purposes, density gradient centrifugation still represents a highly reliable and reproducible technique. This article describes two protocols to purify peroxisomes from either liver tissue or the HepG2 hepatoma cell line. The protocol for liver enables purification of peroxisome fractions with high purity (95%) and is therefore suitable to study low-abundant peroxisomal proteins or analyze their lipid composition, for example. The protocol presented for HepG2 cells is not suitable to gain highly pure peroxisomal fractions but is intended to be used for gradient profiling experiments and allows easier manipulation of the peroxisomal compartment, e.g., by gene knockdown or protein overexpression for functional studies. Both purification methods therefore represent complementary tools to be used to analyze different aspects of peroxisome physiology. Please note that this is an updated version of a protocol, which has been published in a former volume of Methods in Molecular Biology.

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Acknowledgments

We thank all colleagues who donated antibodies used in this work. We would further like to thank D. Türker and Dr. S. Kühl for technical assistance and Dr. R. Carmichael for critical reading of the manuscript.

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Authors and Affiliations

  1. Institute of Neuroanatomy, Medical Faculty Mannheim, Mannheim Centre for Translational Neuroscience, Mannheim, Germany

    Andreas Manner & Markus Islinger

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  1. Andreas Manner
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  2. Markus Islinger
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Correspondence to Markus Islinger .

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Editors and Affiliations

  1. Faculty of Health and Life Sciences, Biosciences, University of Exeter, Exeter, Devon, UK

    Michael Schrader

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Manner, A., Islinger, M. (2023). Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation. In: Schrader, M. (eds) Peroxisomes. Methods in Molecular Biology, vol 2643. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3048-8_1

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  • DOI: https://doi.org/10.1007/978-1-0716-3048-8_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3047-1

  • Online ISBN: 978-1-0716-3048-8

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