This volume details a comprehensive and extensive set of protocols for the study of autophagy in vitro and in vivo. Chapters focus on mammals, various model organisms, and provide protocols for the study of autophagy-related processes outside of the canonical autophagy pathways. 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. Authoritative and…mehr
This volume details a comprehensive and extensive set of protocols for the study of autophagy in vitro and in vivo. Chapters focus on mammals, various model organisms, and provide protocols for the study of autophagy-related processes outside of the canonical autophagy pathways. 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.
Authoritative and cutting-edge, Autophagy: Methods and Protocols aims to ensure successful results in the further study of this vital field.
Recombinant Expression, Purification and Assembly of p62 Filaments.- Structural Studies of Autophagy-related Proteins.- Structural Studies of Mammalian Autophagy Lipidation Complex.- Structural Studies of Selective Autophagy in Yeast.- Biophysical Studies of LC3 Family Proteins.- Reconstituting Autophagy Initiation from Purified Components.- Cell-free Reconstitution of Autophagic Membrane Formation.- Use of Peptide Arrays for Identification and Characterization of LIR Motifs.- Studying Autophagic Lysosome Reformation in Cells and by an invitro Reconstitution System.- Formation of Autophagosomes Coincides With Relaxation of Membrane Curvature.- Studies of Receptor-Atg8 Interactions during Selective Autophagy.- Correlative Light and Electron Miscopy of Autophagosomes.- Improved Electron Microscopy (EM) Fixation Methods for Tracking Autophagy-associated Membranes in Cultured Mammalian Cells.- 3-color Simultaneous Live Imaging of Autophagy-related Structures.- CorrelativeLive-cell Imaging and Super-resolution Microscopy of Autophagy.- Methods for Imaging Autophagosome Dynamics in Primary Neurons.- Imaging Autophagy in hiPSC-derived Midbrain Dopaminergic Neuronal Cultures for Parkinson's Disease Research.- Correlative Light Electron Microscopy to analyze LC3 Proteins in Caenorhabditis elegans embryo.- Imaging Non-canonical Autophagy and LC3-associated Phagocytosis in Cultured Cells.- Measurement of Bulk Autophagy by a Cargo Sequestration Assay.- Methods to Detect Loss of Lysosomal Membrane Integrity.- Monitoring of Autophagy and Cell Volume Regulation in Kidney Epithelial Cells in Response to Fluid Shear Stress.- Identification and Regulation of Multimeric Protein Complexes in Autophagy via SILAC- based Mass Spectrometry Approaches.- Identification and Validation of Novel Autophagy Regulators using an Endogenous Readout siGENOME Screen.- Autophagy Pathway Mapping to Elucidate the Function of Novel Autophagy Regulators Identified by High-throughput Screening.- In vitro Screening Platforms for Identifying Autophagy Modulators in Mammalian Cells.- In vitro Screening Platforms for Identifying Autophagy Modulators in Mammalian Cells.- Methods for the Study of Entotic Cell Death.- MHC Class I Internalization via Autophagy Proteins.- Analysis of Autophagy for Liver Pathogenesis.- Autophagy in 3D In Vitro and Ex Vivo Cancer Models.- Autophagy in Platelets.- Methods to Image Macroautophagy in the Brain In Vivo.- Measuring Nonselective and Selective Autophagy in the Liver.- Measuring Autophagy in Pancreatitis.- Characterization of the "autophagic flux" in Prostate Cancer Tissue Biopsies by LC3A/LAMP2a Immunofluorescence and Confocal Microscopy.- Methods to Determine the Role of Autophagy Proteins in C. elegans Ageing.- Investigating Non-selective Autophagy in Drosophila.- Imaging the Dynamics of Mitophagy in Live Cells.- Triggering Mitophagy with Photosensitizers.- Investigating Mitophagy and Mitochondrial Morphologyin vivo using mito-QC.- Assays to Monitor Mitophagy in Drosophila.- Mitophagy Dynamics in Caenorhabditis elegans.- Methods for Studying Mitophagy in Yeast.- Measuring Anti-bacterial Autophagy.- Quantitative Phosphoproteomics of Selective Autophagy Receptors.- Analysis of Chaperone-mediated Autophagy.- Interactive Autophagy: Monitoring a Novel Form of Selective Autophagy by Macroscopic Observations.
Recombinant Expression, Purification and Assembly of p62 Filaments.- Structural Studies of Autophagy-related Proteins.- Structural Studies of Mammalian Autophagy Lipidation Complex.- Structural Studies of Selective Autophagy in Yeast.- Biophysical Studies of LC3 Family Proteins.- Reconstituting Autophagy Initiation from Purified Components.- Cell-free Reconstitution of Autophagic Membrane Formation.- Use of Peptide Arrays for Identification and Characterization of LIR Motifs.- Studying Autophagic Lysosome Reformation in Cells and by an invitro Reconstitution System.- Formation of Autophagosomes Coincides With Relaxation of Membrane Curvature.- Studies of Receptor-Atg8 Interactions during Selective Autophagy.- Correlative Light and Electron Miscopy of Autophagosomes.- Improved Electron Microscopy (EM) Fixation Methods for Tracking Autophagy-associated Membranes in Cultured Mammalian Cells.- 3-color Simultaneous Live Imaging of Autophagy-related Structures.- CorrelativeLive-cell Imaging and Super-resolution Microscopy of Autophagy.- Methods for Imaging Autophagosome Dynamics in Primary Neurons.- Imaging Autophagy in hiPSC-derived Midbrain Dopaminergic Neuronal Cultures for Parkinson's Disease Research.- Correlative Light Electron Microscopy to analyze LC3 Proteins in Caenorhabditis elegans embryo.- Imaging Non-canonical Autophagy and LC3-associated Phagocytosis in Cultured Cells.- Measurement of Bulk Autophagy by a Cargo Sequestration Assay.- Methods to Detect Loss of Lysosomal Membrane Integrity.- Monitoring of Autophagy and Cell Volume Regulation in Kidney Epithelial Cells in Response to Fluid Shear Stress.- Identification and Regulation of Multimeric Protein Complexes in Autophagy via SILAC- based Mass Spectrometry Approaches.- Identification and Validation of Novel Autophagy Regulators using an Endogenous Readout siGENOME Screen.- Autophagy Pathway Mapping to Elucidate the Function of Novel Autophagy Regulators Identified by High-throughput Screening.- In vitro Screening Platforms for Identifying Autophagy Modulators in Mammalian Cells.- In vitro Screening Platforms for Identifying Autophagy Modulators in Mammalian Cells.- Methods for the Study of Entotic Cell Death.- MHC Class I Internalization via Autophagy Proteins.- Analysis of Autophagy for Liver Pathogenesis.- Autophagy in 3D In Vitro and Ex Vivo Cancer Models.- Autophagy in Platelets.- Methods to Image Macroautophagy in the Brain In Vivo.- Measuring Nonselective and Selective Autophagy in the Liver.- Measuring Autophagy in Pancreatitis.- Characterization of the "autophagic flux" in Prostate Cancer Tissue Biopsies by LC3A/LAMP2a Immunofluorescence and Confocal Microscopy.- Methods to Determine the Role of Autophagy Proteins in C. elegans Ageing.- Investigating Non-selective Autophagy in Drosophila.- Imaging the Dynamics of Mitophagy in Live Cells.- Triggering Mitophagy with Photosensitizers.- Investigating Mitophagy and Mitochondrial Morphologyin vivo using mito-QC.- Assays to Monitor Mitophagy in Drosophila.- Mitophagy Dynamics in Caenorhabditis elegans.- Methods for Studying Mitophagy in Yeast.- Measuring Anti-bacterial Autophagy.- Quantitative Phosphoproteomics of Selective Autophagy Receptors.- Analysis of Chaperone-mediated Autophagy.- Interactive Autophagy: Monitoring a Novel Form of Selective Autophagy by Macroscopic Observations.
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