Peroxisomes, 1st ed. 2017 Methods and Protocols Methods in Molecular Biology Series, Vol. 1595

This volume provides easily accessible and comprehensive collection of methods, techniques, and strategies to investigate the molecular and cellular biology of peroxisomes in different organisms. Chapters detail valuable instructions, guidelines and protocols for molecular cell biologists, biochemists and biomedical researchers with an interest in peroxisome biology. Chapters in Peroxisomes: Methods and Protocols illustrate the isolation of peroxisomes, investigation of properties of membrane proteins, protocols to investigate and manipulate peroxisomes in cellular systems, detection of peroxisomes, including immunofluorescence, cytochemistry, cryo-immuno electron microscopy, and live cell imaging approaches.

Authoritative and practical, Peroxisomes: Methods and Protocols aims to be useful for those already working on peroxisomes as well as for those who would like to start working on this fascinating organelle.

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

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 Labelled 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

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 Immunolabelling of Peroxisomal Proteins

Tina Schrader, Markus Islinger, and Michael Schrader

13. Labelling of Peroxisomes for Live Cell Imaging in the Filamentous Fungus Ustilago Maydis

Sofia C. Guimaraes, 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

Marc Fransen and Chantal Brees

16. Determination of Peroxisomal pH in Living Mammalian Cells Using pHRed

Luis F. Godinho, and Michael Schrader

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

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

25. Flow Cytometric Analysis of the Expression Pattern of Peroxisomal Proteins, Abcd1, Abcd2, and Abcd3 in BV-2 Murine Microglial Cells

Thomas Nury, Imen Helali, El Mostafa Karym, Meryam Debbabi, 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

27. Analysis of Peroxisomal β-Oxidation during Storage Oil Mobilization in Arabidopsis Thaliana Seedlings

Björn Hielscher, Lennart Charton, Tabea Mettler-Altmann, and Nicole Linka

Noa Dahan, Maya Schuldiner, and Einat Zalckvar

29. Generation of Peroxisome-Deficient Somatic Animal Cell Mutants

Kanji Okumoto and Yukio Fujiki

Ronald J.A. Wanders, Femke C.C. Klouwer, Sacha Ferdinandusse, Hans R. Waterham, and Bwee Tien Poll-Thé

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts

Includes supplementary material: sn.pub/extras