Methods in
Molecular Biology 1595

Michael Schrader Editor

Peroxisomes
Methods and Protocols


Methods

in

Molecular Biology

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

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Peroxisomes
Methods and Protocols

Edited by

Michael Schrader
College of Life and Enivornmental Sciences, Biosciences, University of Exeter, Exeter, UK

Editor
Michael Schrader
College of Life and Environmental Sciences, Biosciences
University of Exeter
Exeter, UK

ISSN 1064-3745    ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6935-7    ISBN 978-1-4939-6937-1 (eBook)
DOI 10.1007/978-1-4939-6937-1
Library of Congress Control Number: 2017937360
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This book is dedicated to Tina, Anna and Paula—the
“lighthouses” in my life—who make it all possible
and to my parents for their ongoing support
and interest in peroxisomes.


Preface
This edition of Peroxisomes: Methods and Protocols assembles a volume of easily accessible
protocols particularly useful for those already working on peroxisomes (and other membrane-bound organelles) as well as for those who would like to start working on this fascinating organelle. Due to their growing importance in health and development, there is
increasing interest in the study of peroxisomes. Furthermore, peroxisomes combine properties which render them suitable model organelles to study diverse molecular processes in
eukaryotic cells.
This edition assembles a comprehensive collection of methods, techniques and strategies to investigate the molecular and cellular biology of peroxisomes in different organisms.
It aims to provide valuable instructions, guidelines and protocols for molecular cell biologists, biochemists and biomedical researchers with an interest in peroxisome biology.
Protocols addressing peroxisomes in humans, yeast, fungi and plants are covered.
Chapters illustrating the isolation of peroxisomes, investigation of properties of membrane
proteins, biochemical assays to measure peroxisome metabolic function or protocols to
investigate and manipulate peroxisomes in cellular systems have been included. Other chapters address the detection of peroxisomes, including immunofluorescence, cytochemistry,
cryo-immuno-electron microscopy and live cell imaging approaches. More specialised
chapters deal with peroxisomal redox measurements, determination of pH, peroxisome
biogenesis, import of peroxisomal proteins, protein modification or pexophagy, to name a
few. Finally, the clinical and laboratory diagnosis of peroxisomal disorders and the use of
patient fibroblasts are addressed.
I would like to express my sincerest appreciation to all of the authors who contributed
chapters to this volume. They were a pleasure to work with, providing state-of-the-art protocols (and one review) in a timely fashion, while cheerfully responding to all of my queries.
I would also like to thank Professor John Walker, editor of the Methods in Molecular Biology
series, for his invaluable advice and input in all aspects of the formulation of this book.
This is truly an exciting time to be involved in peroxisome research, as vital functions
of this dynamic organelle in humans, plants and fungi are being discovered. I hope you will
get excited about peroxisome biology, that you will take advantage of the methods, techniques and strategies provided and that this volume of protocols will serve you well to

tackle peroxisome- and organelle-based research questions.
Exeter, Devon, UK

Michael Schrader

vii


Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
  1 Isolation of Peroxisomes from Rat Liver and Cultured Hepatoma
Cells by Density Gradient Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Andreas Manner and Markus Islinger
  2 Isolation of Peroxisomes from Mouse Brain Using a Continuous
Nycodenz Gradient: A Comparison to the Isolation of Liver
and Kidney Peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Miriam J. Schönenberger and Werner J. Kovacs
  3 Determining the Topology of Peroxisomal Proteins Using Protease
Protection Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tânia Francisco, Ana F. Dias, Ana G. Pedrosa, Cláudia P. Grou,
Tony A. Rodrigues, and Jorge E. Azevedo
  4 Isolation of Native Soluble and Membrane-Bound Protein Complexes
from Yeast Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tobias Hansen, Anna Chan, Thomas Schröter, Daniel Schwerter,
Wolfgang Girzalsky, and Ralf Erdmann
  5 Method for Measurement of Peroxisomal Very Long-Chain Fatty
Acid Beta-Oxidation and De Novo C26:0 Synthesis Activity in Living
Cells Using Stable-Isotope Labeled Docosanoic Acid . . . . . . . . . . . . . . . . . . . .
Malu-Clair van de Beek, Inge M.E. Dijkstra, and Stephan Kemp

  6 Analysis of Plasmalogen Synthesis in Cultured Cells . . . . . . . . . . . . . . . . . . . . .
Masanori Honsho and Yukio Fujiki
  7 Transfection of Primary Human Skin Fibroblasts for Peroxisomal Studies . . . . .
Janet Koster and Hans R. Waterham
  8 siRNA-mediated Silencing of Peroxisomal Genes in Mammalian Cells . . . . . . .
Tina A. Schrader and Michael Schrader
  9 Dual Reporter Systems for the Analysis of Translational Readthrough
in Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Julia Hofhuis, Severin Dieterle, Rosemol George, Fabian Schueren,
and Sven Thoms
10 Cytochemical Detection of Peroxisomes in Light and Electron
Microscopy with 3,3′-diaminobenzidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H. Dariush Fahimi
11 Cryo-Immuno Electron Microscopy of Peroxisomal Marker Proteins . . . . . . . .
Karina Mildner and Dagmar Zeuschner
12 Detection and Immunolabeling of Peroxisomal Proteins . . . . . . . . . . . . . . . . .
Tina A. Schrader, Markus Islinger, and Michael Schrader

ix

1

13

27

37

45
55

63
69

81

93
101
113


x

Contents

13 Labeling of Peroxisomes for Live Cell Imaging in the Filamentous
Fungus Ustilago maydis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sofia C. Guimarães, Sreedhar Kilaru, Michael Schrader,
and Martin Schuster
14 Quantitative Monitoring of Subcellular Redox Dynamics in Living
Mammalian Cells Using RoGFP2-Based Probes . . . . . . . . . . . . . . . . . . . . . . . .
Celien Lismont, Paul A. Walton, and Marc Fransen
15 KillerRed as a Tool to Study the Cellular Responses
to Peroxisome-Derived Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Marc Fransen and Chantal Brees
16 Determination of Peroxisomal pH in Living Mammalian Cells
Using pHRed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Luis F. Godinho and Michael Schrader
17 In Cellulo Approaches to Study Peroxisomal Protein Import – Yeast
Immunofluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tobias Hansen, Wolfgang Girzalsky, and Ralf Erdmann

18 Blue Native PAGE: Applications to Study Peroxisome Biogenesis . . . . . . . . . . .
Kanji Okumoto, Shigehiko Tamura, and Yukio Fujiki
19 In Vitro PMP Import Analysis Using Cell-Free Synthesized PMP
and Isolated Peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Yuqiong Liu, Masanori Honsho, and Yukio Fujiki
20 Peroxisomal Membrane and Matrix Protein Import Using a Semi-Intact
Mammalian Cell System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kanji Okumoto, Masanori Honsho, Yuqiong Liu, and Yukio Fujiki
21 The Use of Glycosylation Tags as Reporters for Protein Entry
into the Endoplasmic Reticulum in Yeast and Mammalian Cells . . . . . . . . . . . .
Judith Buentzel and Sven Thoms
22 Detection of Ubiquitinated Peroxisomal Proteins in Yeast . . . . . . . . . . . . . . . .
Natasha Danda and Chris Williams
23 Assessing Pexophagy in Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Shun-ichi Yamashita and Yukio Fujiki
24 Experimental Systems to Study Yeast Pexophagy . . . . . . . . . . . . . . . . . . . . . . .
Shun-ichi Yamashita, Masahide Oku, Yasuyoshi Sakai,
and Yukio Fujiki
25 Flow Cytometric Analysis of the Expression Pattern of Peroxisomal
Proteins, Abcd1, Abcd2, and Abcd3 in BV-2 Murine Microglial Cells . . . . . . .
Meryam Debbabi, Thomas Nury, Imen Helali, El Mostafa Karym,
Flore Geillon, Catherine Gondcaille, Doriane Trompier, Amina Najid,
Sébastien Terreau, Maryem Bezine, Amira Zarrouk, Anne Vejux,
Pierre Andreoletti, Mustapha Cherkaoui-Malki, Stéphane Savary,
and Gérard Lizard
26 Study of Peroxisomal Protein Phosphorylation by Functional Proteomics . . . . .
Andreas Schummer, Sven Fischer, Silke Oeljeklaus,
and Bettina Warscheid

131


151

165

181

191
197

207

213

221
233
243
249

257

267


Contents

27 Analysis of Peroxisomal β-Oxidation During Storage
Oil Mobilization in Arabidopsis thaliana Seedlings . . . . . . . . . . . . . . . . . . . . . .
Björn Hielscher, Lennart Charton, Tabea Mettler-Altmann,
and Nicole Linka

28 Peroxisome Mini-Libraries: Systematic Approaches to Study
Peroxisomes Made Easy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Noa Dahan, Maya Schuldiner, and Einat Zalckvar
29 Generation of Peroxisome-Deficient Somatic Animal Cell Mutants . . . . . . . . . .
Kanji Okumoto and Yukio Fujiki
30 Clinical and Laboratory Diagnosis of Peroxisomal Disorders . . . . . . . . . . . . . .
Ronald J.A. Wanders, Femke C.C. Klouwer, Sacha Ferdinandusse,
Hans R. Waterham, and Bwee Tien Poll-Thé

xi

291

305
319
329

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343


Contributors
Pierre Andreoletti  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de
Bourgogne Franche Comté, Dijon, France
Jorge E. Azevedo  •  Instituto de Investigação e Inovação em Saúde, Universidade do
Porto, Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de
Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal;
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto,
Porto, Portugal
Maryem Bezine  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme
lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne

Franche Comté, Dijon, France; Laboratoire de Venins et Biomolécules Thérapeutiques
(LVMT), Université de Tunis El Manar-Institut Pasteur, Tunis, Tunisia
Chantal Brees  •  Laboratory of Lipid Biochemistry and Protein Interactions,
Department of Cellular and Molecular Medicine, KU Leuven – University of
Leuven, Leuven, Belgium
Judith Buentzel  •  Department of Pediatrics and Adolescent Health, University
Medical Center, University of Göttingen, Göttingen, Germany
Anna Chan  •  Abteilung für Systembiochemie, Institut für Biochemie und
Pathobiochemie, Medizinische Fakultät der Ruhr-­Universität Bochum,
Bochum, Germany
Lennart Charton  •  Institute for Plant Biochemistry and Cluster of Excellence on
Plant Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany
Mustapha Cherkaoui-Malki  •  Laboratoire ‘Biochimie du peroxysome, inflammation
et métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université
de Bourgogne Franche Comté, Dijon, France
Noa Dahan  •  Department of Molecular Genetics, Weizmann Institute of Science,
Rehovot, Israel
Natasha Danda  •  Molecular Cell Biology, Groningen Biomolecular Sciences and
Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands
Meryam Debbabi  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de
Bourgogne Franche Comté, Dijon, France; Faculté de Médecine, Laboratoire de
Nutrition—Aliments Fonctionnels et Santé Vasculaire (LR12ES05), Monastir &
Faculté de Médecine, Université de Monastir, Sousse, Tunisia
Ana F. Dias  •  Instituto de Investigação e Inovação em Saúde, Universidade do Porto,
Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de Biologia
Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal;
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto,
Porto, Portugal

xiii



xiv

Contributors

Severin Dieterle  •  Department of Pediatrics and Adolescent Health, University
Medical Center Göttingen, University of Göttingen, Göttingen, Germany
Inge M.E. Dijkstra  •  Laboratory Genetic Metabolic Diseases (F0-226),
Departments of Pediatrics and Clinical Chemistry, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Ralf Erdmann  •  Abteilung für Systembiochemie, Institut für Biochemie und
Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum,
Ruhr-Universität Bochum, Bochum, Germany
H. Dariush Fahimi  •  Division of Medical Cell Biology, Department of Anatomy
and Cell Biology, University of Heidelberg, Heidelberg, Germany
Sacha Ferdinandusse  •  Laboratory Genetic Metabolic Diseases, Departments of
Paediatrics and Clinical Chemistry, Emma Children’s Hospital, Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands
Sven Fischer  •  Department of Biochemistry and Functional Proteomics, Institute of
Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany
Tânia Francisco  •  Instituto de Investigação e Inovação em Saúde, Universidade do
Porto, Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de
Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal
Marc Fransen  •  Laboratory of Lipid Biochemistry and Protein Interactions,
Department of Cellular and Molecular Medicine, University of Leuven - KU Leuven,
Leuven, Belgium
Yukio Fujiki  •  Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
Flore Geillon  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme
lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne

Franche Comté, Dijon, France
Rosemol George  •  Department of Pediatrics and Adolescent Health, University
Medical Center Göttingen, Georg August University Göttingen, Göttingen, Germany
Wolfgang Girzalsky  •  Abteilung für Systembiochemie, Institut für Biochemie und
Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, Bochum,
Germany
Luis F. Godinho  •  Department of Medical Sciences and Institute for Biomedicine
(iBiMED), University of Aveiro, Aveiro, Portugal
Catherine Gondcaille  •  Laboratoire ‘Biochimie du peroxysome, inflammation et
métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université
de Bourgogne Franche Comté, Dijon, France
Cláudia P. Grou  •  Instituto de Investigação e Inovação em Saúde, Universidade do
Porto, Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de
Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal
Sofia C. Guimarães  •  College of Life and Environmental Sciences, Biosciences,
University of Exeter, Exeter, UK
Tobias Hansen  •  Abteilung für Systembiochemie, Institut für Biochemie und
Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, Bochum,
Germany
Imen Helali  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme
lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne
Franche Comté, Dijon, France; Faculté de Pharmacie, Laboratoire des Maladies


Contributors

xv

Transmissibles et Substances Biologiquement Actives (LR99ES27), Université de
Monastir, Monastir, Tunisia

Björn Hielscher  •  Institute for Plant Biochemistry and Cluster of Excellence on Plant
Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany
Julia Hofhuis  •  Department of Pediatrics and Adolescent Health, University Medical
Center Göttingen, University of Göttingen, Göttingen, Germany
Masanori Honsho  •  Medical Institute of Bioregulation, Kyushu University, Fukuoka,
Japan
Markus Islinger  •  Center for Biomedicine and Medical Technology Mannheim,
Institute of Neuroanatomy, University of Heidelberg, Mannheim, Germany
EL Mostafa Karym  •  Laboratoire ‘Biochimie du peroxysome, inflammation et
métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université
de Bourgogne Franche Comté, Dijon, France; Laboratoire de Biochimie et
Neuroscience, Faculté de Sciences et Techniques, Université Hassan 1er, Settat,
Morocco
Stephan Kemp  •  Laboratory Genetic Metabolic Diseases (F0-226), Departments of
Pediatrics and Clinical Chemistry, Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands
Sreedhar Kilaru  •  College of Life and Environmental Sciences, Biosciences, University
of Exeter, Exeter, UK
Femke C.C. Klouwer  •  Laboratory Genetic Metabolic Diseases, Departments of
Paediatrics and Clinical Chemistry, Emma Children’s Hospital, Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands
Janet Koster  •  Laboratory Genetic Metabolic Diseases, Department of Clinical
Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The
Netherlands
Werner J. Kovacs  •  Institute of Molecular Health Sciences, ETH Zurich, Zurich,
Switzerland
Nicole Linka  •  Institute for Plant Biochemistry and Cluster of Excellence on Plant
Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany
Celien Lismont  •  Laboratory of Lipid Biochemistry and Protein Interactions,
Department of Cellular and Molecular Medicine, University of Leuven - KU Leuven,

Leuven, Belgium
Yuqiong Liu  •  Graduate School of Systems Life Sciences, Kyushu University Graduate
School, Fukuoka, Japan
Gérard Lizard  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme
lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne
Franche Comté, Dijon, France
Andreas Manner  •  Institute of Neuroanatomy, Center for Biomedicine and Medical
Technology Mannheim, University of Heidelberg, Mannheim, Germany
Tabea Mettler-Altmann  •  Institute for Plant Biochemistry and Cluster of Excellence
on Plant Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany
Karina Mildner  •  Max-Planck-Institute for Molecular Biomedicine, Electron
Microscopy, Muenster, Germany


xvi

Contributors

Amina Najid  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme
lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne
Franche Comté, Dijon, France
Thomas Nury  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme
lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne
Franche Comté, Dijon, France
Silke Oeljeklaus  •  Department of Biochemistry and Functional Proteomics, Institute
of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany
Masahide Oku  •  Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Kyoto, Japan
Kanji Okumoto  •  Department of Biology, Faculty of Sciences, Kyushu University,
Fukuoka, Japan

Ana G. Pedrosa  •  Instituto de Investigação e Inovação em Saúde, Universidade do
Porto, Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de
Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal;
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto,
Porto, Portugal
Bwee Tien Poll-Thé  •  Department Paediatric Neurology, Emma Children’s Hospital,
Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Tony A. Rodrigues  •  Instituto de Investigação e Inovação em Saúde, Universidade do
Porto, Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de
Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal;
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto,
Porto, Portugal
Yasuyoshi Sakai  •  Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Kyoto, Japan
Stephane Savary  •  Laboratoire ‘Biochimie du peroxysome, inflammation et
métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université
de Bourgogne Franche Comté, Dijon, France
Miriam J. Schönenberger  •  Institute of Physiology, University of Zurich, Zurich,
Switzerland
Michael Schrader  •  College of Life and Environmental Sciences, Biosciences,
University of Exeter, Exeter, UK
Tina A. Schrader  •  College of Life and Environmental Sciences, Biosciences,
University of Exeter, Exeter, UK
Thomas Schröter  •  Abteilung für Systembiochemie, Institut für Biochemie und
Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, Bochum,
Germany
Fabian Schueren  •  Department of Pediatrics and Adolescent Health, University
Medical Center Göttingen, Georg August University Göttingen, Göttingen, Germany
Maya Schuldiner  •  Department of Molecular Genetics, Weizmann Institute of Science,
Rehovot, Israel

Andreas Schummer  •  Department of Biochemistry and Functional Proteomics,
Institute of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany
Martin Schuster  •  College of Life and Environmental Sciences, Biosciences,
University of Exeter, Exeter, UK


Contributors

xvii

Daniel Schwerter  •  Abteilung für Systembiochemie, Institut für Biochemie und
Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, Bochum,
Germany
Shigehiko Tamura  •  Division for Experimental Natural Science, Faculty of Arts
and Science, Kyushu University, Fukuoka, Japan
Sebastien Terreau  •  Laboratoire ‘Biochimie du peroxysome, inflammation et
métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université
de Bourgogne Franche Comté, Dijon, France
Sven Thoms  •  Department of Pediatrics and Adolescent Health, University Medical
Center, University of Göttingen, Göttingen, Germany
Doriane Trompier  •  Laboratoire ‘Biochimie du peroxysome, inflammation et
métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université
de Bourgogne Franche Comté, Dijon, France
Malu-Clair van De Beek  •  Laboratory Genetic Metabolic Diseases (F0-226),
Departments of Pediatrics and Clinical Chemistry, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Anne Vejux  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme
lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne
Franche Comté, Dijon, France
Paul A. Walton  •  Laboratory of Lipid Biochemistry and Protein Interactions,

Department of Cellular and Molecular Medicine, University of Leuven - KU Leuven,
Leuven, Belgium; Department of Anatomy and Cell Biology, University of Western
Ontario, London, Canada
Ronald J.A. Wanders  •  Laboratory Genetic Metabolic Diseases, Departments of
Paediatric and Clinical Chemistry, Emma Children’s Hospital, Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands
Bettina Warscheid  •  Department of Biochemistry and Functional Proteomics,
Institute of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany;
BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg,
Germany
Hans R. Waterham  •  Laboratory Genetic Metabolic Diseases, Department of Clinical
Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The
Netherlands
Chris Williams  •  Molecular Cell Biology, Groningen Biomolecular Sciences and
Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands
Shun-Ichi Yamashita  •  Department of Cellular Physiology, Graduate School of
Medical and Dental Sciences, Niigata University, Niigata, Japan
Einat Zalckvar  •  Department of Molecular Genetics, Weizmann Institute of Science,
Rehovot, Israel
Amira Zarrouk  •  Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme
lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne
Franche Comté, Dijon, France; Faculté de Médecine, Laboratoire de Nutrition –
Aliments Fonctionnels et Santé Vasculaire, Monastir & Faculté de Médecine,
Université de Monastir, Sousse, Tunisia
Dagmar Zeuschner  •  Max-Planck-Institute for Molecular Biomedicine, Electron
Microscopy, Muenster, Germany


Chapter 1
Isolation of Peroxisomes from Rat Liver and Cultured

Hepatoma Cells by Density Gradient Centrifugation
Andreas Manner and Markus Islinger
Abstract
Subcellular fractionation is still a valuable technique to unravel organelle-specific proteomes, validate the
location of uncharacterized proteins, or to functionally analyze import and metabolism in individual subcellular compartments. In this respect, density gradient centrifugation still represents a very classic, indispensable technique to isolate and analyze peroxisomes. Here, we present two independent protocols for
the purification of peroxisomes from either liver tissue or the HepG2 hepatoma cell line. While the former
permits the isolation of highly pure peroxisomes suitable for, e.g., subcellular proteomics experiments, the
latter protocol yields peroxisomal fractions from considerably less purity but allows to easily modify metabolic conditions in the culture medium or to genetically manipulate the peroxisomal compartment. In this
respect, both purification methods represent alternative tools to be applied in experiments investigating
peroxisome physiology.
Key words Peroxisomes, Liver, Density gradient centrifugation, Organelle purification, ACAD11

1  Introduction
Peroxisomes are ubiquitous organelles, which can be found in differing amounts in all eukaryotic cells. Morphologically, peroxisomes appear in varying sizes and shapes in different tissues and
contain a differing subset of proteins. Accordingly, there is no standard procedure for peroxisome isolation and protocols have to be
adapted to the tissue of interest. In mammals, highest amounts of
peroxisomes can be found in hepatocytes and the kidney cells of
the proximal tubule, where they can comprise up to 2% of the total
cellular content. In these organs peroxisomes do not only stand
out by their sheer amount but also organelle size and can reach
diameters of up to 1 μm. Accordingly, peroxisomes from liver and
kidney possess unique physical features that enable the isolation of
highly pure peroxisome fractions that can still not be obtained in
this quality in other tissues. Also historically, the liver of rats were
the source of the fractions used for the initial biochemical
Michael Schrader (ed.), Peroxisomes: Methods and Protocols, Methods in Molecular Biology, vol. 1595,
DOI 10.1007/978-1-4939-6937-1_1, © Springer Science+Business Media LLC 2017

1



2

Andreas Manner and Markus Islinger

c­ haracterization of peroxisomes by De Duve and colleagues [1, 2].
This initial protocol, as well as most of the methods applied to
date, is based on a three-step isolation procedure consisting of (1)
a mild homogenization of the tissue using a Potter-Elvenheijm
homogenizer at low velocities, (2) a series of differential centrifugations leading to a peroxisome-enriched fraction, and (3) a final
centrifugation step applying a density gradient. For the fundamental experiments performed by the De Duve group, linear sucrose
density gradients were used for isopycnic centrifugation; however,
they required the intravenous injection of Triton WR-1339 prior
to sacrificing the animals. In addition, the isolation of peroxisomes
of high purity required the use of a special Beaufay-type rotor
allowing loading and unloading during centrifugation [3].
Metrizamide, a tri-iodinated benzamido-derivative of glucose is—
compared to sucrose—a considerably less viscous gradient medium
showing lower osmolality and is not able to penetrate biological
membranes [4]. Its use as a gradient medium allowed the isolation
of peroxisomes with purities >90% from rodent liver and kidney
without the use of a special pretreatment or rotor inventory [5–7].
Metrizamide was subsequently replaced by the iodinated non-glucose-based benzamido-derivatives Nycodenz and Optiprep, which
are more stable, less toxic, and show significantly less interference
with biological compounds [8, 9]. Since then, both media have
been frequently used to isolate peroxisomes from rodent liver or
kidney and have been shown to yield peroxisome fractions of high
purity suitable for subsequent proteome analysis without further
affinity purification steps [10, 11]. While peroxisomes sediment in
isoosmotic solutions at velocities overlapping with small mitochondria, lysosomes, and partially microsomes, they reach unexpectedly

high densities, when centrifuged in gradients from the iodinated
media mentioned above. This remarkable behavior may be due to
the selective permeability of peroxisomes to the gradient media.
Interestingly, peroxisomes possess comparably large protein pores
allowing the free diffusion of molecules up to 600 Da across their
membrane [12]. The molecular sizes of Metrizamide, Nycodenz,
and Optiprep range between 821 and 1550 Da, respectively, and
should not allow those molecules to enter peroxisomes through
these pores. A more likely explanation may lie in the high fragility
of peroxisomes during fractionation procedures [13]: Osmotic
damage during the isolation may result in a transient disruption of
the peroxisomal membrane enabling the exchange of soluble compounds. Thus, the lower osmotic pressure in gradient media from
higher molecular weight compounds would lead to a better preservation of and reduced uptake of separation medium by the organelles. Indeed, liver peroxisomes sediment in Metrizamide at a mean
density of 1.245 compared to 1.195 in Optiprep [5, 14], ­suggesting
that the bulkier Optiprep penetrates the organelle membrane less
efficiently.


Peroxisome Isolation from Liver and Hepatoma Cells

3

Since peroxisomes from liver and kidney have been thoroughly
characterized in numerous publications one may ask if a chapter
describing the isolation of peroxisomes from these tissues is still
required in a current method compilation. However, as the purities
of these peroxisome fractions still exceed those from other tissues
and cell culture, they still remain the gold-standard for the localization of newly identified, ubiquitously expressed peroxisomal proteins. Still, some peroxisomal constituents may only localize at the
organelle under specific physiological conditions or show tissuespecific expression. In this respect, cell cultures represent ideal
models to manipulate metabolic conditions by administration of

selected compounds or to modify peroxisomal functions by knockdown or overexpression of distinct peroxisomal proteins. Hence,
peroxisomes isolated from cell lines may be used to allocate novel
endogenous or overexpressed peroxisomal proteins that do not permanently associate with the organelle or which under standard conditions are only present at very low concentrations. HepG2 cells
possess a considerable number of peroxisomes and according to
their liver origin express the proteins of the classic peroxisomal metabolic pathways. Thus, they can be easily analyzed using commercially available antibodies against peroxisomal marker proteins.
Peroxisomes in this cell line are on average considerably smaller
than those from rodent liver or kidney and the majority of peroxisomes sediments at densities that are closer to the endoplasmic
reticulum and mitochondria [15]. Accordingly, peroxisomal fractions from HepG2 cells do not reach comparable purities but are
nevertheless suitable to allocate individual proteins to distinct
organelle fractions. To this end, we established a protocol to separate peroxisomes from HepG2 cells from the remaining organelles
using a flat, linear density gradient. As both isolation schemes (from
liver and HepG2 cells) represent multi-step procedures, representative flow-charts are shown as an overview in Fig. 1 and may assist to
compare the methods used for isolating peroxisomes from cells and
tissues. As an example for a localization experiment, we show here
the distribution of one of the more recently identified peroxisomal
constituents, the acyl-CoA dehydrogenase ACAD11 [10, 11, 16,
17] in the subcellular fractions from liver and HepG2 cells.

2  Materials
2.1  General
Materials

1.Refractometer for the preparation of density gradients.
2. Ultracentrifuge and a fixed angle rotor (e.g., VTi 50, VTi 65.1
type vertical rotor, Beckman Coulter, Brea, USA).
3.Homogenization buffer (HB): 250 mM sucrose, 5 mM
MOPS, 1 mM EDTA, 2 mM PMSF, 1 mM DTT, 1 mM
ɛ-aminocaproic acid, pH 7.4 adjusted with KOH.



4

Andreas Manner and Markus Islinger

Fig. 1 Schematic overview of peroxisome isolation from rodent liver (a) and HepG2 cells (b). PNS post nuclear
supernatant, HM heavy mitochondrial fraction, LM light mitochondrial fraction, MIC microsomal fraction, CYT
cytosolic fraction

4.Gradient buffer (GB): 5 mM MOPS, 1 mM EDTA, 2 mM
PMSF, 1 mM DTT, 1 mM ɛ-aminocaproic acid, pH 7.4
adjusted with KOH.
2.2  Isolation of Rat
Liver Peroxisomes

1. Motor-driven Potter-Elvehjem tissue grinder with loose fitting
pestle (clearance 0.1–0.15 mm, vol. 30 mL).
2.Optiprep: 60% (w/v) iodixanol solution in water (Axis Shield,
Rodeløkka, Sweden).
3.Quick-seal polyallomer tubes 25 × 89 mm (39 mL, Beckman
Coulter).
4.0.9% (w/v) NaCl solution.

2.3  Separation
of Peroxisomes
from HepG2 Cells

1.Gradient mixer (10–25 mL volume per chamber).
2.Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM
KCl, 1.8 mM KH2PO4, 10 mM Na2HPO4.
3.Cell scrapers to remove HepG2 cells from culture dishes.

4.Syringe (5 mL) with a 27G needle.
5. Quick-seal polyallomer tubes 16 × 76 mm (13.5 mL, Beckman
Coulter).


Peroxisome Isolation from Liver and Hepatoma Cells

5

3  Methods
3.1  Isolation
of Peroxisomes
from Rat Liver

1.To produce the sigmoid-shaped density gradient prepare
Optiprep solutions of 1.12, 1.15, 1.19, 1.22, and 1.26 g/mL
by diluting the 60% Optiprep stock solution (1.32 g/mL) with
GB. To adjust the correct density use a refractometer and the
formula:
ρ = 3.350 × refractive index − 3.462.
2.Using the 39 mL Quick-Seal tubes (Beckman Coulter Inc.),
layer sequentially 4, 3, 6, 7, and 10 mL of the Optiprep dilutions
in a decreasing order of concentration (1.26–1.12 g/mL).
3.Freeze the discontinuous gradient rapidly in liquid nitrogen
and store at −80 °C (see Note 1).
4.Precool all solutions and vessels used on ice; all subsequent
purification steps are carried out at a temperature of 4 °C.
5. After anesthesia open the body cavity of the animals and excise
the liver, rinse with 0.9% NaCl and determine its weight (see
Note 2). Cut the liver into small pieces and wash away drained

blood with HB. Finally suspend liver pieces in ice-cold HB at a
ratio of 3 mL/g tissue (see Note 3).
6.Homogenize the liver pieces with the Potter-Elvehjem tissue
grinder at 1000 rpm using only one stroke in 2 min (see Note 4).
7.Centrifuge the homogenate at 600 × gav for 10 min, 4 °C to
pellet cellular debris and nuclei.
8.Keep the supernatant on ice, resuspend the pellet in approx.
10 mL and re-homogenize the pellet for a second time at
1000 rpm using one stroke of 1 min. Repeat the 600 × gav
centrifugation step.
9.Pool the supernatants from both homogenization steps and
centrifuge at 2700 × gmax for 10 min, 4 °C to pellet the heavy
mitochondrial fraction, which mainly contains large
mitochondria.
10.Carefully drain the pellet from the supernatant and manually
suspend the pellet in HB using a glass rod. Make sure not to
disturb the blood pellet at the bottom of the tube.
11.Repeat the 2700 × gmax centrifugation step.
12.Combine the supernatants from both runs and centrifuge at
37,000 × gmax for 20 min at 4 °C. This run will produce the
pellet of light mitochondria, which contains peroxisomes.
They are enriched by the factor of 3–4 if compared to the PNS,
combined with microsomes, mitochondria, and lysosomes.
13.Remove the supernatant (see Note 5) and then carefully aspirate the gel-like, reddish “fluffy” layer positioned at the top of
the pellet, which mainly contains microsomes.


6

Andreas Manner and Markus Islinger


14.Stir the remaining dry pellet with a glass rod until no clumps
are visible and subsequently add drop-wise HB. Continue stirring until you obtain a homogenous organelle suspension.
Adjust volume of HB to at least 2 mL/g of pellet.
15.Wash the light mitochondrial pellet using another centrifugation step at 37,000 × g, 15 min, 4 °C.
16.Again remove the remaining fluffy-layer and resuspend the
residual pellet in 1–2 mL HB/g as described above. This suspension comprises the final light mitochondrial fraction, which
is further separated by the density gradient centrifugation.
17.Slowly defrost the Optiprep gradient described above at room
temperature in a metal stand. This takes around 30 min and
can be initiated in parallel to the last steps of the differential
centrifugation procedure.
18.Layer 5 mL of the light mitochondrial fraction on the top of
the Optiprep gradient. Overlay with GB and seal the tubes (see
Note 6).
19.Centrifuge in a vertical rotor (e.g., VTi50) at an integrated
force of 1256 × 106 × g × min (gmax = 33,000) with slow acceleration/deceleration, 4 °C.
20.After centrifugation, three narrow but clearly detectable bands
will be visible near the bottom of the tube. The lowermost is composed of crystalloid cores from disrupted peroxisomes. Somewhat
above you will detect two bands containing intact peroxisomes.
The lower band at the higher density of 1.20 g/mL is the purest
fraction in the gradient containing more than 95% of peroxisomes. The one above at 1.18 g/mL shows a higher contamination with mitochondria but still contains usually above 90% of
peroxisomes. To collect the individual fractions, puncture the
tubes with a syringe and aspirate band by band.
21.To concentrate the samples and wash out the Optiprep, dilute
the sample at least 3:1 in HB. Pellet the organelles by centrifugation at 37,000 × gmax and resuspend in an appropriate amount
of HB.
22.Determine enzyme activities of organelle marker enzymes as
described [18] (or chapters this issue). Alternatively, immunoblots can be performed as shown in Fig. 2.
3.2  Purification

of Peroxisomes
from HepG2 Cells

1.Remove HepG2 cells from minimum five 80% confluent
75 cm2 cell culture flasks using a cell scraper (for immunoblot
analysis 10–12 flasks are recommended). Clear cells from culture medium by centrifugation at 500 × gmax, 5 min. Wash in
10 mL PBS by repeating the centrifugation procedure.


Peroxisome Isolation from Liver and Hepatoma Cells

7

Fig. 2 Peroxisome isolation from rodent liver. (a) Sketch of a typical sigmoid Optiprep-gradient after centrifugation.
Organelles enrich in characteristic bands at densities given to the right, arrow—location where fraction LM is
layered onto the gradient. (b) Corresponding immunoblots representing the distribution of different organelle
marker proteins. Note that peroxisomes enrich in two individual bands, LM1 and LM2. While the LM1 fraction
usually possesses a purity above 95%, LM2 show a higher contamination with mitochondria, which can, however,
be significantly reduced by repetition of the gradient centrifugation [14]. Note that ACAD11, which was recently
reported to localize to mitochondria in neuronal cells [19], is clearly enriched in the peroxisomal fraction of rat
liver tissue [17]. (c) Comassie-stained 12.5% SDS-gel of the fractions used for the immunoblots depicted in (b).
PNS post nuclear supernatant, HM heavy mitochondrial fraction, LM light mitochondrial fraction, MIC microsomal
fraction, CYT cytosolic fraction, Cor fraction of crystalloid cores from disrupted peroxisomes. Antibodies: rabbit α
ACAD11 (1:2000, gift from J. Vockley, University of Pittsburgh), rabbit α ACOX1 (1:10,000, gift from T. Hashimoto,
Shinshu University School of Medicine, Nagano), mouse α ABCD3 (1:5000, Sigma-­Aldrich), rabbit α Pex14
(1:10,000, gift from D. Crane, Griffith University, Brisbane), rabbit α Catalase (1:10,000, gift from A. Völkl, University
of Heidelberg), mouse α ECI2 (1:1000, BD Biosciences), rabbit α UOX (Uricase 1:100, gift from A. Völkl, University
of Heidelberg), mouse α VDAC1 (1:1000, Abcam), mouse α ATP synthase α (1:30,000, BD Biosciences), mouse α
PDI (1:1000, Abcam), mouse α GRP78 (1:1000, BD Biosciences)



8

Andreas Manner and Markus Islinger

2.Resuspend the cell pellet in 2 mL HB/tissue culture flask and
disrupt the cells by shearing through a syringe with a 27G needle for seven times.
3.Collect undisrupted cells, cellular debris, and nuclei by centrifugation at 600 × gmax, 10 min, 4 °C. Keep the supernatant
on ice until further use. Homogenize the pellet for a second
time as described above and centrifuge again at 600 × g,
10 min, 4 °C.
4.Pool both post nuclear supernatants (PNS) and centrifuge at
2000 × gav for 15 min at 4 °C.
5.Decant the supernatant and centrifuge at 20,000 × gav to produce the light mitochondrial pellet (LM). Resuspend the pellet
in 1 mL of HB using a glass rod.
6.For the next centrifugation step, pour a linear Nykodenz-­
gradient ranging from 1.14 to 1.20 g/mL immediately before
use by applying a gradient mixer (see Note 7). To pour the
gradient, prepare two stock solutions of Nycodenz of 1.14 and
1.19 g/mL in GB, pH 7.4. You will require 6 mL of each
Nykodenz solution per 16 × 76 mm tube (e.g., 13.5 mL
Quick-Seal tubes, Beckman Coulter Inc.).
7.Layer the pellet suspended in 1 mL HB on the top of the
Nycodenz gradient. Overlay with GB and seal the tubes.
Centrifuge at 100,000 × g for 3 h, 4 °C.
8.As the individual bands in the gradient are usually very faint,
puncture the tube at the bottom with a syringe and retrieve
equal-sized samples (e.g., 1 mL).
9.To remove the Nycodenz and enrich organelles, dilute the
samples at minimum 3:1 in HB and centrifuge at >30,000 × g

for 20 min, 4 °C. Suspend the resulting pellets in a small
amount of HB and store at −80 °C until further analysis.
10.Perform immunoblots or enzyme assays (see above) to evaluate the separation (see Fig. 3 as an example) (see Note 8).

4  Notes
1. Freezing of the density step-gradient used in the liver isolation
protocol is a prerequisite for a successful separation. The characteristic sigmoid density distribution is generated during the
thawing process.
2. Glycogen deposits in the liver will disturb the separation in the
density gradient. Thus, to obtain highly pure peroxisome fractions the animals have to be fasted overnight.
3.The protocol for liver peroxisomes can also be applied for the
isolation of peroxisomes from kidney. If comparable amounts


Peroxisome Isolation from Liver and Hepatoma Cells

9

Fig. 3 Peroxisome isolation from HepG2 cells. (a) Sketch of the linear gradient (1.14–1.19 g/mL) used for the
organelle separation from HepG2 cells. Positions of the individual fractions analyzed in the neighboring immunoblot and their correspondent densities after the centrifugation step are depicted. The arrow represents the
position, where the LM fraction was layered onto the gradient. (b) Immunoblot showing the distribution of
mitochondria, endoplasmic reticulum, and peroxisomes in the gradient. Maxima of the individual organelle
peaks are marked by arrowheads. Note that ACAD11 is not associated with mitochondria in HepG2 cells (see
also [17] and correspondent immunofluorescence in chapter (c)). However, the ACAD11 peak fraction slightly
differs from the maxima of the peroxisome markers ACBD3, ACOX1, and Pex14. Since ACAD11 expression is
highly dynamic in HepG2 cells, this observation may point to a specific peroxisome subfraction containing
ACAD11. PNS post nuclear supernatant, HM heavy mitochondrial fraction, LM light mitochondrial fraction.
Antibodies: see legend of Fig. 2 for ACAD11, ACOX1, ABCD3, Pex14, ATP synthase α, PDI and GRP78; mouse α
COX4 (1:2000, Abcam)


of starting material are used, however, the peroxisome yield
will be lower than in liver. As kidney peroxisomes do not contain uricase (UOX), the separation will produce no core fraction, which is a characteristic for liver peroxisomes.
4.Peroxisomes are particularly fragile and leaky organelles; thus
vigorous homogenization using multiple pestle strokes should
be avoided to maximize organelle integrity.
5.Optionally the supernatant produced while pelleting the light
mitochondrial fraction can be further separated into a
microsome-­enriched pellet and a supernatant representing the
cytosol. These fractions can be used to evaluate the subcellular
location of a protein of interest. To obtain both fractions, add
another centrifugation step at 100,000 × gmax, 30 min, 4 °C.
6.As the thumb-rule for efficient peroxisome purification, the
maximum amount of LM fraction, which can be applied on
one density gradient, equals approximately 10 g of liver tissue
used as a starting material.
7.Adaptors to fit the small 16 mm diameter tubes into a VTi 50
or comparable rotor with a larger cavity are commercially
available.


10

Andreas Manner and Markus Islinger

8.Compared to the situation in the density gradients from liver,
microsomes migrate to a significantly higher density in the linear Nykodenz-gradients, switching the position with mitochondria. This might be due to the different methods used for
homogenization, which may fractionate the tubular ER cisternae to a different degree.

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.
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