Peroxisomes Methods and Protocols 2nd Edition by Michael Schrader ISBN 9781071630471 1071630474

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ISBN 10: 1071630474
ISBN 13: 9781071630471
Author: Michael Schrader

This fully updated volume assembles a comprehensive collection of methods, techniques, and strategies to investigate the molecular and cellular biology of peroxisomes in different organisms. Peroxisome research is on the rise, as novel functions and proteins of this dynamic organelle are still being discovered through studies in model systems including humans, mice, flies, plants, fungi, and yeast, and this progress is reflected in the chapters included in this collection. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and up-to-date, Peroxisomes: Methods and Protocols, Second Edition serves as an ideal guide for researchers working on peroxisome- and organelle-based research questions.

Peroxisomes Methods and Protocols 2nd Edition Table of contents:

Chapter 1: Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation
1 Introduction
2 Materials
2.1 General Materials
2.2 Isolation of Rat Liver Peroxisomes
2.3 Separation of Peroxisomes from HepG2 Cells
3 Methods
3.1 Isolation of Peroxisomes from Rat Liver
3.2 Purification of Peroxisomes from HepG2 Cells
4 Notes
References
Chapter 2: Analysis of Yeast Peroxisomes via Spatial Proteomics
1 Introduction
2 Materials
2.1 Yeast and Culture Media
2.2 Cell Lysis and Preparation of Spheroplasts
2.3 Subcellular Fractionation
2.3.1 Differential Centrifugation
2.3.2 Nycodenz Density Gradient Centrifugation
2.4 LC-MS Sample Preparation
2.4.1 Acetone Precipitation
2.4.2 Proteolytic In-Solution Digestion
2.4.3 Desalting of Peptides
2.5 LC-MS/MS Analysis
2.6 Data Analysis
3 Methods
3.1 Yeast Cell Culture and Harvest
3.2 Cell Lysis and Generation of Spheroplasts
3.3 Subcellular Fractionation
3.3.1 Preparation of Cellular Fractions by Differential Centrifugation
3.3.2 Generation of a Gradient-Purified Peroxisomal Fraction by Nycodenz Density Gradient Centrifugation
3.4 LC-MS Sample Preparation
3.4.1 Acetone Precipitation
3.4.2 Proteolytic In-Solution Digestion
3.4.3 Desalting of Peptides and Preparation of Samples for LC-MS Analysis
3.5 LC-MS Analysis
3.6 Data Analysis
4 Notes
References
Chapter 3: Isolation of Glycosomes from Trypanosoma brucei
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Reagents and Solutions
2.3 Materials
3 Methods
3.1 Cultivation of T. brucei Cells and Harvesting
3.2 Rupture of the Cells with Silicon Carbide
3.3 Preparation of an Organelle Enriched Fraction by Differential Centrifugation
3.4 Isolation of Glycosomes by Density Gradient Centrifugation
3.5 Successive Enrichment of Glycosomal Membrane Proteins
4 Notes
References
Chapter 4: Immunolabeling for Detection of Endogenous and Overexpressed Peroxisomal Proteins in Mammalian Cells
1 Introduction
2 Materials
2.1 Mammalian Cells and Plasmids
2.2 Cell Culture Equipment
2.3 Cell Culture Media, Buffers, and Reagents
2.4 Transfection
2.5 Immunofluorescence and Fluorescence Microscopy
2.6 Controls
2.7 Antibody Sources
3 Methods
3.1 Cell Culture
3.2 Transfection of Human Skin Fibroblasts (dMFF) Using Microporation (Optional)
3.3 Detection of Expressed (or Endogenous) Peroxisomal Proteins
4 Notes
References
Chapter 5: Super-Resolution Imaging of Peroxisomal Proteins Using STED Nanoscopy
1 Introduction
2 Materials
2.1 Mammalian Cells and Culturing
2.1.1 Media and Buffers
2.1.2 Equipment
2.2 Yeast Cells and Cultivation
2.2.1 Media and Buffers
2.2.2 Equipment
2.3 (Live-Cell) Dyes and Antibodies (See Notes 5 and 6)
2.4 Fixed Sample Preparation for Confocal or STED Nanoscopy
2.5 STED Nanoscopy
3 Methods
3.1 Seeding HEK Cells for Immunofluorescence
3.2 Immunofluorescence of Fixed HEK Cells for STED Nanoscopy
3.3 HaloTag or SNAP-Tag Labeling of Mammalian Cells for STED Nanoscopy
3.3.1 Live-Cell Labeling of HEK Cells
3.3.2 Fixed-Cell Labeling of HEK Cells with Live Dyes
3.4 HaloTag or SNAP-Tag Labeling of Fixed Yeast Cells for STED Nanoscopy
3.5 STED Nanoscopy
4 Notes
References
Chapter 6: Direct Stochastic Optical Reconstruction Microscopy (dSTORM) of Peroxisomes
1 Introduction
2 Materials
2.1 Immunofluorescence.
2.2 Super-Resolution Imaging
3 Methods
3.1 Immunofluorescence
3.2 Super-Resolution Imaging
4 Notes
References
Chapter 7: Correlative Light- and Electron Microscopy in Peroxisome Research
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Equipment
2.3 Software
3 Methods
3.1 Fixing the Yeast Cells
3.2 Gelatin Embedding and Cryo-Protection
3.3 Preparation of Gold Decorated Grids
3.4 Cryo-Sectioning
3.5 Fluorescence Microscopy
3.6 Transmission Electron Microscopy and Image Alignment
4 Notes
References
Chapter 8: Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 Mammalian Cells
2.1.2 Cell Culture Equipment
2.1.3 Cell Culture Media and Reagents
2.2 Processing and Embedding of Cultured Cells for TEM
2.3 Ultrathin Sectioning and Contrasting of TEM Sections
2.4 TEM Imaging and Stereological Analysis
3 Methods
3.1 Cell Culture
3.2 Primary Fixation of Cell Monolayers
3.3 Sample Processing and Embedding
3.4 Ultramicrotomy and Contrasting of TEM Sections
3.5 TEM Analysis and Sampling Procedures
3.6 Spatial Stereology
4 Notes
References
Chapter 9: Detection of Peroxisomal Proteins During Mycobacterial Infection
1 Introduction
2 Materials
2.1 Culturing Mycobacterium sp.
2.1.1 Solid and Liquid Culturing Media
2.1.2 Consumables
2.1.3 Instruments
2.2 Cell Culture
2.2.1 Immunofluorescence Microscopy
3 Methods
3.1 Culturing of Mycobacterium sp.
3.1.1 Preparation of Middlebrook 7H9 Liquid Culture Media
3.1.2 Culturing M. tb in Middlebrook 7H9 Liquid Culture Media
Safety Precautions
Culture Methods
3.1.3 Preparation of Solid Middlebrook 7H11 Agar Plates
3.1.4 Culturing M. tb on Solid Mycobacteria 7H11 Agar Plates
3.1.5 Preparation of M. tb Glycerol Stocks
3.2 Immunofluorescence Microscopy
3.2.1 Isolation of Bone Marrow-Derived Macrophages (BMDMs)
3.2.2 Seeding of Macrophages
Calculation of Seeding of BMDMs
3.2.3 Bacterial Infection
3.2.4 Microscopic Slide Preparation
4 Notes
References
Chapter 10: Proximity-Ligation Assay to Detect Peroxisome-Organelle Interaction
1 Introduction
2 Materials
2.1 Mammalian Cells and Plasmids
2.2 Cell Culture Equipment
2.3 Cell Culture Media, Buffers, and Reagents
2.4 Transfection
2.5 Proximity Ligation Assay (PLA)
2.6 Controls
2.7 Primary Antibodies
3 Methods
3.1 Cell Culture
3.2 DEAE-Dextran Transfection of COS-7 cells (see Notes 14 and 15)
3.3 Detection of Protein Proximity by PLA
3.4 Quantification of Peroxisome-Organelle/ER Contacts
4 Notes
References
Chapter 11: Assay of Reactive Oxygen/Nitrogen Species (ROS/RNS) in Arabidopsis Peroxisomes Through Fluorescent Protein Contain…
1 Introduction
2 Materials
2.1 Plant Samples
2.2 Fluorescent Probes
2.3 Confocal Laser Scanning Microscopy (CLSM)
3 Methods
4 Notes
References
Chapter 12: Identification of Peroxisome-Derived Hydrogen Peroxide-Sensitive Target Proteins Using a YAP1C-Based Genetic Probe
1 Introduction
2 Materials
2.1 Proteomics Sample Preparation
2.1.1 Equipment
2.1.2 Compounds and Materials for Keratin Removal from Surfaces and Objects
2.1.3 Other Materials
2.1.4 Compounds and Buffers
2.2 Cell Culture and Cell Treatment
2.3 Electrophoretic Mobility Shift Assay and Immunoblotting
2.4 LC-MS/MS
3 Methods
3.1 Measures to Reduce Keratin Contamination
3.2 Cell Culture
3.2.1 Maintenance of the Cells
3.2.2 Pretreatment of po-DD-DAO Flp-In T-REx 293 Cells
3.3 Sample Preparation
3.3.1 Preparation of Cell Suspension
3.3.2 Cell Treatment
3.3.3 Preparation of a Cleared Cell Lysate
3.3.4 Enrichment of IBD-SBP-YAP1C and IBD-SBP-YAP1C-Trapped Proteins on a Streptavidin Affinity Matrix
3.3.5 Removal of the Non-bound Proteins from the Streptavidin Affinity Matrix
3.3.6 Elution of the IBD-SBP-YAP1C-Trapped Proteins
3.3.7 Reduction and Alkylation of Available Thiol Groups
3.3.8 Protein Precipitation
3.3.9 Trypsin Digestion
3.3.10 Peptide Desalting
3.4 Quality Control of the Samples
3.5 Identification of IBD-SBP-YAP1C-Trapped Proteins Using LC-MS/MS
3.6 Data Analysis
4 Notes
References
Chapter 13: Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein …
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 Equipment
2.1.2 Materials
2.2 Electroporation
2.3 Fluorescence Microscopy
3 Methods
3.1 Cell Culture
3.2 Neon Electroporation of Flp-In T-REx 293 Cells
3.2.1 Preparation of the Cells
3.2.2 Electroporation of the Cells
3.3 Pyridine Nucleotide Measurements and Analysis
3.3.1 Preparing for Live-Cell Imaging
3.3.2 Image Collection
3.3.3 Image Analysis
3.4 Functional Validation of po-SoNar
3.5 Functional Validation of po-iNAP1
4 Notes
References
Chapter 14: Live-Cell Imaging of Peroxisomal Calcium Levels and Dynamics
1 Introduction
2 Materials
2.1 Materials for Ca2+ Measurement
2.2 Setup for Live-Cell Imaging
3 Methods
3.1 Cell Culture and Cell Preparation
3.2 Measurement of Ca2+ in HeLa or Any Other Histamine-Responsive Cells
3.3 Measurement of Ca2+ in Histamine-Insensitive (or Histamine-Sensitive) Cells
3.4 Data Analysis for FRET Ca2+ Sensors
3.5 Calibration Experiments for Absolute Ca2+ Concentration
3.6 Determination of the Dissociation Constant Kd
3.7 Calculation of Absolute Ca2+ Concentration
4 Notes
References
Chapter 15: Analysis of Peroxisome Biogenesis by Phos-Tag SDS-PAGE
1 Introduction
2 Materials
2.1 SDS Polyacrylamide Gel Containing Phos-Tag
2.2 Transfer to PVDF Membrane
2.3 Sample Preparation
2.4 Cell Culture and Immunoblotting
2.5 Equipment
3 Methods
3.1 Casting of Phos-Tag PAGE Gel
3.2 Sample Preparation (from Cultured Cells)
3.3 Electrophoresis
3.4 Transfer to PVDF Membrane
4 Notes
References

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Tags: Michael Schrader, Peroxisomes Methods, Protocols