Paul Pryor

Subcellular Fractionation: A Laboratory Manual Paul Pryor

Marc Notes: Includes bibliographical references and index. Table of Contents: Acknowledgments CHAPTER 1 IntroductionIntroduction Paul R. Pryor CHAPTER 2 Lectin-Magnetic Beads for Plasma Membrane IsolationPROTOCOLLectin-Magnetic Beads for Plasma Membrane Isolation Yu-Chen Lee, Hsuan-Chen Liu, Carol Chuang, and Sue-Hwa Lin CHAPTER 3 Isolation of Clathrin-Coated Vesicles from Tissue Culture CellsINTRODUCTIONIsolation of Clathrin-Coated Vesicles from Tissue Culture Cells Georg H. H. Borner and Andrew B. FieldingPROTOCOLS1 Isolating HeLa Cell Fractions Enriched for Clathrin-Coated Vesicles Georg H. H. Borner and Andrew B. Fielding2 Using In-Gel Digestion and an Orbitrap Mass Spectrometer to Analyzethe Proteome of Clathrin-Coated Vesicles Georg H. H. Borner and Andrew B. Fielding3 Using In-Solution Digestion, Peptide Fractionation, and a Q Exactive MassSpectrometer to Analyze the Proteome of Clathrin-Coated Vesicles Georg H. H. Borner and Andrew B. Fielding CHAPTER 4 Purification of Early and Late EndosomesINTRODUCTIONPurification of Early and Late Endosomes Mariana E. G. Arau jo, Giorgia Lamberti, and Lukas A. HuberPROTOCOLS1 Homogenization of Mammalian Cells Mariana E. G. Arau jo, Giorgia Lamberti, and Lukas A. Huber2 Isolation of Early and Late Endosomes by Density Gradient Centrifugation Mariana E. G. Arau jo, Giorgia Lamberti, and Lukas A. Huber3 Isolation of Macrophage Early and Late Endosomes by Latex Bead Internalizationand Density Gradient Centrifugation Giorgia Lamberti, Mariana E. G. Arau jo, and Lukas A. Huber CHAPTER 5 The Synaptosome as a Model System for Studying Synaptic PhysiologyINTRODUCTIONThe Synaptosome as a Model System for Studying Synaptic Physiology Gareth J. O. EvansPROTOCOL1 Subcellular Fractionation of the Brain: Preparation of Synaptosomesand Synaptic Vesicles Gareth J. O. Evans CHAPTER 6 Isolating Phagosomes from Tissue Culture CellsPROTOCOLIsolating Phagosomes from Tissue Culture Cells Paul R. Pryor and Adam P. Rofe CHAPTER 7 Strategies for Isolation of ExosomesINTRODUCTIONStrategies for Isolation of Exosomes Emily Zeringer, Timothy Barta, Mu Li, and Alexander V. Vlassov CHAPTER 8 Isolation of Lysosomes from Rat Tissue and Tissue Culture CellsINTRODUCTIONIsolation of Lysosomes from Rat Tissue and Tissue Culture Cells Paul R. PryorPROTOCOLS1 Isolating Lysosomes from Rat Liver Paul R. Pryor2 Purification of Lysosomes Using Supraparamagnetic Iron OxideNanoparticles (SPIONs) Adam P. Rofe and Paul R. Pryor CHAPTER 9 Isolation of Specialized Secretory Compartments: GLUT4 Storage VesiclesINTRODUCTIONIsolation of Specialized Secretory Compartments: GLUT4 Storage Vesicles Jessica B. A. Sadler, Christopher A. Lamb, Gwyn W. Gould, and Nia J. BryantPROTOCOLS1 Preparation of a Total Membrane Fraction from 3T3-L1 Adipocytes Jessica B. A. Sadler, Christopher A. Lamb, Gwyn W. Gould, and Nia J. Bryant2 16K Fractionation of 3T3-L1 Adipocytes to Produce a Crude GLUT4-ContainingVesicle Fraction Jessica B. A. Sadler, Christopher A. Lamb, Gwyn W. Gould, and Nia J. Bryant3 Complete Membrane Fractionation of 3T3-L1 Adipocytes Jessica B. A. Sadler, Christopher A. Lamb, Gwyn W. Gould, and Nia J. Bryant4 Iodixanol Gradient Centrifugation to Separate Components of the Low-DensityMembrane Fraction from 3T3-L1 Adipocytes Jessica B. A. Sadler, Christopher A. Lamb, Gwyn W. Gould, and Nia J. Bryant CHAPTER 10 Isolation of Peroxisomes from YeastINTRODUCTIONIsolation of Peroxisomes from Yeast Jana Cramer, Daniel Effelsberg, Wolfgang Girzalsky, and Ralf ErdmannPROTOCOLS1 Small-Scale Purification of Peroxisomes for Analytical Applications Jana Cramer, Daniel Effelsberg, Wolfgang Girzalsky, and Ralf Erdmann2 Large-Scale Purification of Peroxisomes for Preparative Applications Jana Cramer, Daniel Effelsberg, Wolfgang Girzalsky, and Ralf Erdmann CHAPTER 11 Isolation and Analysis of Microtubules and Associated ProteinsINTRODUCTIONIsolation and Analysis of Microtubules and Associated Proteins Roger D. SlobodaPROTOCOLS1 Isolation of Microtubules by Assembly/Disassembly Methods Roger D. Sloboda2 Isolation of Microtubules and Microtubule-Associated ProteinsUsing Paclitaxel Roger D. Sloboda3 Separation of Tubulin and Microtubule-Associated Proteins by Ion ExchangeChromatography Roger D. Sloboda4 Isolation of Microtubule-Based Motor Proteins by ATP Release fromPaxlitaxel-Stabilized Microtubules Roger D. Sloboda5 Observation of Microtubule-Based Motor Protein Activity Roger D. Sloboda CHAPTER 12 Golgi IsolationPROTOCOLGolgi Isolation Danming Tang and Yanzhuang Wang CHAPTER 13 Isolation of Mitochondria from Cells and TissuesINTRODUCTIONIsolation of Mitochondria from Cells and Tissues David A. Clayton and Gerald S. ShadelPROTOCOLS1 Isolation of Mitochondria from Tissue Culture Cells David A. Clayton and Gerald S. Shadel2 Isolation of Mitochondria from Animal Tissue David A. Clayton and Gerald S. Shadel3 Purification of Mitochondria by Sucrose Step Density GradientCentrifugation David A. Clayton and Gerald S. Shadel CHAPTER 14 Isolation of Chloroplasts from Plant ProtoplastsPROTOCOL1 Isolation of Chloroplasts from Plant Protoplasts Shiu-Cheung Lung, Matthew D. Smith, and Simon D. X. Chuong CHAPTER 15 Subcellular Fractionation of Rough MicrosomesINTRODUCTIONSubcellular Fractionation of Rough Microsomes David D. SabatiniPROTOCOLS1 Preparation of Rough Microsomes from Rat LiverDavid D. Sabatini2 Preparation of Crude Rough Microsomes from Dog Pancreas David D. Sabatini3 Preparation of Crude Rough Microsomes from Tissue Culture Cells David D. Sabatini CHAPTER 16 Purification of Ribosomes, Ribosomal Subunits, and PolysomesINTRODUCTIONPurification of Ribosomes, Ribosomal Subunits, and Polysomes Maria C. Rivera, Bruce Maguire, and James A. LakePROTOCOLS1 Isolation of Ribosomes and Polysomes Maria C. Rivera, Bruce Maguire, and James A. Lake2 Isolation of Ribosomes by Chromatography Bruce A. Maguire3 Purification of 70S RibosomesMaria C. Rivera, Bruce Maguire, and James A. Lake4 Purification of Polysomes Maria C. Rivera, Bruce Maguire, and James A. Lake5 Dissociation of Ribosomes into Large and Small Subunits Maria C. Rivera, Bruce Maguire, and James A. Lake CHAPTER 17 Isolation of NucleiINTRODUCTIONIsolation of Nuclei Arash Nabbi and Karl RiabowolPROTOCOLS1 Rapid Isolation of Nuclei from Cells In Vitro Arash Nabbi and Karl Riabowol2 Isolation of Pure Nuclei Using a Sucrose Cushion Arash Nabbi and Karl Riabowol CHAPTER 18 The Nuclear Matrix: Fractionation Techniques and AnalysisINTRODUCTIONThe Nuclear Matrix: Fractionation Techniques and Analysis Rosemary H. C. Wilson, Emma L. Hesketh, and Dawn CoverleyPROTOCOL1 Preparation of the Nuclear Matrix for Parallel Microscopy andBiochemical Analyses Rosemary H. C. Wilson, Emma L. Hesketh, and Dawn Coverley APPENDIX General Safety and Hazardous Material Information Index"Biographical Note: Paul Pryor is a newly appointed lecturer in the Centre for Immunology and Infection (CII) and HYMS. Paul obtained his BSc in Biochemistry from the University of York and then went on to do a PhD in Cell Biology in the laboratory of Professor Geoff Holman, University of Bath. Paul then undertook post-doctoral research in the laboratory of Professor J. Paul Luzio, University of Cambridge, researching the molecular mechanisms in delivery of material to lysosomes. In 2005 Paul was awarded a Wellcome Trust Career Development Fellowship and moved to the Department of Biology (York) in 2007, investigating phagolysosome biogenesis. Publisher Marketing: Eukaryotic cells are remarkably complex structures, containing a vast repertoire of macromolecules, organelles, and other compartments that orchestrate the tasks required for life. For in-depth studies of their function and composition, reliable methods for the isolation of specific subcellular structures are often required. This laboratory manual provides step-by-step protocols for the extraction of subcellular components from animal tissues, yeasts, plants, and cultured cells. Each chapter focuses on a particular eukaryotic organelle, vesicle, membrane, or macromolecular complex. Strategies for breaking cells while maintaining the structural and functional integrity of the component of interest, enriching for that component based on its physical and biochemical characteristics, and monitoring and ensuring the success of the purification procedure are provided. The contributors describe both traditional approaches (e.g., density gradient centrifugation) and innovative techniques (e.g., the use of SPIONs) for isolating subcellular constituents. This manual is therefore an essential laboratory resource for all cell biologists seeking a comprehensive collection of dependable subcellular fractionation methods. Contributor Bio:  Pryor, Paul born 1839