This volume provides readers with a collection of new and classical methods, techniques, and applications used to address enduring questions about the structure and functions of the Golgi complex. The chapters in this volume cover diverse topics ranging from model systems; live and fixed cell imaging techniques; in vitro biochemical reconstitution systems; and specific methods developed to study Golgi formation, maintenance, and functions under physiological and pathological conditions. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions…mehr
This volume provides readers with a collection of new and classical methods, techniques, and applications used to address enduring questions about the structure and functions of the Golgi complex. The chapters in this volume cover diverse topics ranging from model systems; live and fixed cell imaging techniques; in vitro biochemical reconstitution systems; and specific methods developed to study Golgi formation, maintenance, and functions under physiological and pathological conditions. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions 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.
Cutting-edge and authoritative, Golgi: Methods and Protocols is a valuable tool for researchers in the field who wish to explore new areas of Golgi biology and for new investigators interested in exploring Golgi structure and function.
Live Cell Imaging of Yeast Golgi Dynamics.- Quantification of Golgi Protein Mislocalization to the Budding Yeast Vacuole.- Immunofluorescence as a Method to Study Golgi Organization in Larval Salivary Glands of Drosophila melanogaster.- Microscopy and Immunocytochemistry-Based Methods to Study Cell Wall Biosynthetic Enzymes in the Golgi.- Reconstitution of Golgi Biogenesis in Permeabilized Trypanosoma brucei Cells.- Generation of GM130 Conditional Knockout Mouse.- Monitoring Effects of Membrane Traffic via Changes in Cell Polarity and Morphogenesis in Three-Dimensional Human Pluripotent Stem Cell Cysts.- Immunofluorescence Microscopy of the Mammalian Golgi Apparatus.- Studying the Organization of the Golgi by Super-Resolution Microscopy.- Super-Resolution Live Imaging of Cargo Traffic Through the Golgi Apparatus in Mammalian Cells.- Studying Golgi Structure and Function by Thin Section TEM.- Algorithm for Modern Electron Microscopic Examination of the Golgi Complex.- High-Pressure Freezing Followed by Freeze-Substitution, An Optimal Electron Microscope Technique to Study Golgi Apparatus Organization and Membrane Trafficking.- Negative Staining Electron Microscopy for Morpho-Functional Characterization of Purified Golgi Membranes from Mammalian Cells.- An Electron Tomographic Analysis of Giantin Deficient Golgi Proposes a New Function of the Golgin Protein Family.- Modeling the Cryo-EM Structure of the Exocyst Complex.- Quantitatively Assessing Co-Localization of Golgi Proteins by Distance Analysis Using the DiAna Software.- A Primer on Deep Learning-Based Cellular Image Classification of Changes in the Spatial Distribution of the Golgi Apparatus After Experimental Manipulation.- Reconstituting Vesicle Budding from the TGN and Immunoisolation of Vesicles Enriched with a Specific Cargo Client.- Common Assays in Mammalian Golgi Studies.- In Vitro Methods to Investigate the Disassembly of the Golgi Ribbon during the G2-M Transition of the Cell Cycle.- Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines.- Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System.- Generation of Stable Cell Lines Expressing Golgi Reassembly Stacking Proteins (GRASPs) by Viral Transduction.- Quantitative Proteomics Analysis of Purified Rat Liver Golgi.- Comparative Genomics for Evolutionary Cell Biology using AMOEBAE: Understanding the Golgi and Beyond.- Common Markers and Small Molecule Inhibitors in Golgi Studies.- Visualizing Reversible Cisternal Stacking in Budding Yeast Pichia pastoris.- Methods for Studying Membrane-Proximal GAP Activity on Prenylated Rab GTPase Substrates.- Studying the Role of Lipid Geometry in COPI Vesicle Formation.- Unbiased Quantification of Golgi Scattering and Golgi-Centrosome Association.- Detection and Analysis of Microtubule Nucleator Gamma-Tubulin Ring Complex.- Quantification of Golgi Entry and Exit Kinetics of Protein Cargoes.- CARTS Formation Assay.- Quantification of Protein Exit at the Trans-Golgi Network.- Endosomal Transport to Lysosomes and the Trans-Golgi Network in Neurons and Other Cells: Visualizing Maturational Flux.- Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles.- Live imaging of Golgi Outposts in Drosophila Dendritic Arbors.- Targeted Protein Unfolding at the Golgi Apparatus.- Modeling N-Glycosylation: A Systems Biology Approach for Evaluating Changes in the Steady State Organization of Golgi Resident Proteins.- Simple N-Glycan Profile Analysis Using Lectin Staining, Mass Spectrometry, and GlycoMaple.- In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis.- Analysis of Golgi Protein Acetylation using In Vitro Assays and Parallel Reaction Monitoring Mass Spectrometry.- Identification and Characterization of GRASP55 O-GlcNAcylation.- Assaying Sterol-Regulated ER-to-Golgi Transport of SREBP Cleavage-Activating Protein Using Immunofluorescence Microscopy.- Analysis of Golgi Morphology Using Immunofluorescence and CellProfiler Software.- Analysis of Golgi Secretory Functions in Cancer.
Live Cell Imaging of Yeast Golgi Dynamics.- Quantification of Golgi Protein Mislocalization to the Budding Yeast Vacuole.- Immunofluorescence as a Method to Study Golgi Organization in Larval Salivary Glands of Drosophila melanogaster.- Microscopy and Immunocytochemistry-Based Methods to Study Cell Wall Biosynthetic Enzymes in the Golgi.- Reconstitution of Golgi Biogenesis in Permeabilized Trypanosoma brucei Cells.- Generation of GM130 Conditional Knockout Mouse.- Monitoring Effects of Membrane Traffic via Changes in Cell Polarity and Morphogenesis in Three-Dimensional Human Pluripotent Stem Cell Cysts.- Immunofluorescence Microscopy of the Mammalian Golgi Apparatus.- Studying the Organization of the Golgi by Super-Resolution Microscopy.- Super-Resolution Live Imaging of Cargo Traffic Through the Golgi Apparatus in Mammalian Cells.- Studying Golgi Structure and Function by Thin Section TEM.- Algorithm for Modern Electron Microscopic Examination of the Golgi Complex.- High-Pressure Freezing Followed by Freeze-Substitution, An Optimal Electron Microscope Technique to Study Golgi Apparatus Organization and Membrane Trafficking.- Negative Staining Electron Microscopy for Morpho-Functional Characterization of Purified Golgi Membranes from Mammalian Cells.- An Electron Tomographic Analysis of Giantin Deficient Golgi Proposes a New Function of the Golgin Protein Family.- Modeling the Cryo-EM Structure of the Exocyst Complex.- Quantitatively Assessing Co-Localization of Golgi Proteins by Distance Analysis Using the DiAna Software.- A Primer on Deep Learning-Based Cellular Image Classification of Changes in the Spatial Distribution of the Golgi Apparatus After Experimental Manipulation.- Reconstituting Vesicle Budding from the TGN and Immunoisolation of Vesicles Enriched with a Specific Cargo Client.- Common Assays in Mammalian Golgi Studies.- In Vitro Methods to Investigate the Disassembly of the Golgi Ribbon during the G2-M Transition of the Cell Cycle.- Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines.- Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System.- Generation of Stable Cell Lines Expressing Golgi Reassembly Stacking Proteins (GRASPs) by Viral Transduction.- Quantitative Proteomics Analysis of Purified Rat Liver Golgi.- Comparative Genomics for Evolutionary Cell Biology using AMOEBAE: Understanding the Golgi and Beyond.- Common Markers and Small Molecule Inhibitors in Golgi Studies.- Visualizing Reversible Cisternal Stacking in Budding Yeast Pichia pastoris.- Methods for Studying Membrane-Proximal GAP Activity on Prenylated Rab GTPase Substrates.- Studying the Role of Lipid Geometry in COPI Vesicle Formation.- Unbiased Quantification of Golgi Scattering and Golgi-Centrosome Association.- Detection and Analysis of Microtubule Nucleator Gamma-Tubulin Ring Complex.- Quantification of Golgi Entry and Exit Kinetics of Protein Cargoes.- CARTS Formation Assay.- Quantification of Protein Exit at the Trans-Golgi Network.- Endosomal Transport to Lysosomes and the Trans-Golgi Network in Neurons and Other Cells: Visualizing Maturational Flux.- Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles.- Live imaging of Golgi Outposts in Drosophila Dendritic Arbors.- Targeted Protein Unfolding at the Golgi Apparatus.- Modeling N-Glycosylation: A Systems Biology Approach for Evaluating Changes in the Steady State Organization of Golgi Resident Proteins.- Simple N-Glycan Profile Analysis Using Lectin Staining, Mass Spectrometry, and GlycoMaple.- In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis.- Analysis of Golgi Protein Acetylation using In Vitro Assays and Parallel Reaction Monitoring Mass Spectrometry.- Identification and Characterization of GRASP55 O-GlcNAcylation.- Assaying Sterol-Regulated ER-to-Golgi Transport of SREBP Cleavage-Activating Protein Using Immunofluorescence Microscopy.- Analysis of Golgi Morphology Using Immunofluorescence and CellProfiler Software.- Analysis of Golgi Secretory Functions in Cancer.
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