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Full Description
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.
Contents
Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation.- Analysis of Yeast Peroxisomes via Spatial Proteomics.- Isolation of Glycosomes from Trypanosoma brucei.- Immunolabeling for Detection of Endogenous and Overexpressed Peroxisomal Proteins in Mammalian Cells.- Super-Resolution Imaging of Peroxisomal Proteins Using STED Nanoscopy.- Direct Stochastic Optical Reconstruction Microscopy (dSTORM) of Peroxisomes.- Correlative Light- and Electron Microscopy in Peroxisome Research.- Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts.- Detection of Peroxisomal Proteins during Mycobacterial Infection.- Proximity-Ligation Assay to Detect Peroxisome-Organelle Interaction.- Assay of Reactive Oxygen/Nitrogen Species (ROS/RNS) in Arabidopsis Peroxisomes through Fluorescent Protein Containing a Type 1-Peroxisomal Targeting Signal (PTS1).- Identification of Peroxisome-Derived Hydrogen Peroxide-Sensitive Target Proteins Using a YAP1C-Based GeneticProbe.- Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein Sensors.- Live-Cell Imaging of Peroxisomal Calcium Levels and Dynamics.- Analysis of Peroxisome Biogenesis by Phos-Tag SDS-PAGE.- Targeted Modifications of the Yeast Genome to Study Peroxisomes.- Applying CRISPR-Cas9 Genome Editing to Study Genes Involved in Peroxisome Biogenesis or Peroxisomal Functions.- Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins.- Peroxisomes and Viruses: Overview on Current Knowledge and Experimental Approaches.- Tools to Investigate the Peroxisome-Dependent Antiviral Response.- Determining the Importance of Peroxisomal Proteins for Viral Infections in Cultured Mammalian Cells.- Characterization of Yeast Peroxisomes: Enrichment of Peroxisomal Fractions and Analysis of b-Oxidation Activity.- A Cell-Free In Vitro Import System for Peroxisomal Proteins Containing a Type 2 Targeting Signal (PTS2).- Assessing Peroxisomal Protein Interaction by Immunoprecipitation.- Purification of a Recombinant Human PEX1/PEX6 AAA+ ATPase Complex from HEK293TT Cells.- Affinity Purification of Soluble and Membrane-Bound Protein Complexes by a FlpIn Strategy.- Utilization of Nonstop mRNA to Assess Ribosome-Associated Nascent Polypeptide Chains in Early Topogenesis of Peroxisomal Proteins.- Computational Evaluation of Peroxisomal Targeting Signals in Metazoa.- Computational Approaches for Peroxisomal Protein Localization.- Estimating the Interaction Strength between PTS1-Peptides and Their Receptor PEX5 in Living Cells Using Flow-Cytometer-Based FRET (flowFRET) Measurements.- Using the Super Folder GFP (sfGFP) System to Study Plant Peroxisomal Protein Import.- Biochemical Fractionation of Trypanosomes for the Analysis of Glycosomal Protein Import Defects.- Manipulation and Visualization of Peroxisomes in Drosophila.- Mouse Models to Study Peroxisomal Functions and Disorders: Overview,Caveats, and Recommendations.
