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Full Description
This detailed collection gathers both established and recent technical procedures to study the Endosomal Sorting Complex Required for Transport (ESCRT) complexes in a wide range of biological systems: Archaea, A. thaliana, U. maydis, S. cerevisiae, S. pombe, C. elegans, D. melanogaster, and mammalian cells. Opening with a section on imaging techniques, the book continues with chapters covering biochemical approaches presenting strategies for production and characterization of recombinant ESCRT proteins, or of specific ESCRT protein domains, as well as genetic and proteomic experimental approaches. Written for the highly successful Methods in Molecular Biology series, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical,The ESCRT Complexes: Methods and Protocols serves as a compact guide for researchers interested in establishing an integrated approach to investigate the ESCRT machinery functions in cell biology.
Contents
Analysis of the Archaeal ESCRT Apparatus.- Genetic and Cell Biology Methods to Study ESCRTs in Drosophila melanogaster.- Functional Analysis of ESCRT-Positive Extracellular Vesicles in the Drosophila Wing Imaginal Disc.- Subcellular Localization of ESCRT-II in the Nematode C. elegans by Correlative-Light Electron Microscopy.- Proximity Ligation Assay (PLA) to Determine the Endosomal Localization of ESCRT Subunit in Virus-Infected Cells.- Immuno-Localization of ESCRT Proteins in Virus-Infected Cells by Fluorescence and Electron Microscopy.- Single Cell Fluorescence Ratio Image Analysis for Studying ESCRT Function in Receptor Trafficking.- Genetic and Biochemical Analyses of Yeast ESCRT.- Live Imaging of ESCRT Proteins in Microfluidically-Isolated Hippocampal Axons.- Studying the Spatial Organization of ESCRTs in Cytokinetic Abscission Using the High Resolution Imaging Techniques SIM and Cryo-SXT.- Three Dimensional Surface Rendering of ESCRT Proteins Microscopy Data UsingUCSF Chimera Software.- Transient Expression of ESCRT-Components in Arabidopsis Root Cell Suspension Culture-Derived Protoplasts.- Crystallization and Biophysical Approaches for Studying the Interactions between the Vps4-MIT Domain and ESCRT-III Proteins.- Biochemical Approaches to Studying C. elegans ESCRT Function In Vitro.- Purification of Recombinant ESCRT-III Proteins and Their Use in Atomic Force Microscopy and In Vitro Binding and Phosphorylation Assays.- Assessment of ESCRT Protein CHMP5 Activity on Client Protein Ubiquitination by Immunoprecipitation and Western Blotting.- Purification of Plant ESCRT Proteins for Polyclonal Antibody Production.- Genetic and Cytological Methods to Study ESCRT Cell Cycle Function in Fission Yeast.- ESCRT Mutant Analysis and Imaging of ESCRT Components in the Model Fungus Ustilago maydis.- Genetic Suppressor Screen Using an Inducible FREE1-RNAi Line to Detect ESCRT Genetic Interactors in Arabidopsis thaliana.- Screening of Interactions with the ESCRT Machinery by a Gaussia princeps Split Luciferase-Based Complementation Assay.- RNA Interference-Mediated Inhibition of ESCRT in Mammalian Cells.
