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Subcellular Fractionation: A Laboratory Manual


Subject Area(s):  Cell BiologyBiochemistry

By Paul Pryor, Hull York Medical School

Download Free Excerpts from Subcellular Fractionation:

Contents
Introduction: An overview of techniques and key considerations for the separation and isolation of cellular components
Index
Sample Protocol: Isolation of Mitochondria from Tissue Culture Cells


© 2015 • 260 pages, illustrated (6 color, 11 B&W), appendix, references, index
Paperback • £56.00 (click here to price in US Dollars)
ISBN  978-1-621820-42-0

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  •     Description    
  •     Contents    

Description

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) as well as 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.

Contents

Acknowledgments
 
CHAPTER 1 Introduction
Introduction
Paul R. Pryor
 
CHAPTER 2 Lectin-Magnetic Beads for Plasma Membrane Isolation
PROTOCOL
Lectin-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 Cells
INTRODUCTION
Isolation of Clathrin-Coated Vesicles from Tissue Culture Cells
Georg H.H. Borner and Andrew B. Fielding
PROTOCOLS
1 Isolating HeLa Cell Fractions Enriched for Clathrin-Coated Vesicles
Georg H.H. Borner and Andrew B. Fielding
2 Using In-Gel Digestion and an Orbitrap Mass Spectrometer to Analyze the Proteome of Clathrin-Coated Vesicles
Georg H.H. Borner and Andrew B. Fielding
3 Using In-Solution Digestion, Peptide Fractionation, and a Q Exactive Mass Spectrometer to Analyze the Proteome of Clathrin-Coated Vesicles
Georg H.H. Borner and Andrew B. Fielding
 
CHAPTER 4 Purification of Early and Late Endosomes
INTRODUCTION
Purification of Early and Late Endosomes
Mariana E.G. Araújo, Giorgia Lamberti, and Lukas A. Huber
PROTOCOLS
1 Homogenization of Mammalian Cells
Mariana E.G. Araújo, Giorgia Lamberti, and Lukas A. Huber
2 Isolation of Early and Late Endosomes by Density Gradient Centrifugation
Mariana E.G. Araújo, Giorgia Lamberti, and Lukas A. Huber
3 Isolation of Macrophage Early and Late Endosomes by Latex Bead Internalization and Density Gradient Centrifugation
Giorgia Lamberti, Mariana E.G. Araújo, and Lukas A. Huber
 
CHAPTER 5 The Synaptosome as a Model System for Studying Synaptic Physiology
INTRODUCTION
The Synaptosome as a Model System for Studying Synaptic Physiology
Gareth J.O. Evans
PROTOCOL
1 Subcellular Fractionation of the Brain: Preparation of Synaptosomes and Synaptic Vesicles
Gareth J.O. Evans
 
CHAPTER 6 Isolating Phagosomes from Tissue Culture Cells
PROTOCOL
Isolating Phagosomes from Tissue Culture Cells
Paul R. Pryor and Adam P. Rofe
 
CHAPTER 7 Strategies for Isolation of Exosomes
INTRODUCTION
Strategies 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 Cells
INTRODUCTION
Isolation of Lysosomes from Rat Tissue and Tissue Culture Cells
Paul R. Pryor
PROTOCOLS
1 Isolating Lysosomes from Rat Liver
Paul R. Pryor
2 Purification of Lysosomes Using Supraparamagnetic Iron Oxide Nanoparticles (SPIONs)
Adam P. Rofe and Paul R. Pryor
 
CHAPTER 9 Isolation of Specialized Secretory Compartments: GLUT4 Storage Vesicles
INTRODUCTION
Isolation of Specialized Secretory Compartments: GLUT4 Storage Vesicles
Jessica B.A. Sadler, Christopher A. Lamb, Gwyn W. Gould, and Nia J. Bryant
PROTOCOLS
1 Preparation of a Total Membrane Fraction from 3T3-L1 Adipocytes
Jessica B.A. Sadler, Christopher A. Lamb, Gwyn W. Gould, and Nia J. Bryant
2 16K Fractionation of 3T3-L1 Adipocytes to Produce a Crude GLUT4-Containing Vesicle Fraction
Jessica B.A. Sadler, Christopher A. Lamb, Gwyn W. Gould, and Nia J. Bryant
3 Complete Membrane Fractionation of 3T3-L1 Adipocytes
Jessica B.A. Sadler, Christopher A. Lamb, Gwyn W. Gould, and Nia J. Bryant
4 Iodixanol Gradient Centrifugation to Separate Components of the Low-Density Membrane 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 Yeast
INTRODUCTION
Isolation of Peroxisomes from Yeast
Jana Cramer, Daniel Effelsberg, Wolfgang Girzalsky, and Ralf Erdmann
PROTOCOLS
1 Small-Scale Purification of Peroxisomes for Analytical Applications
Jana Cramer, Daniel Effelsberg, Wolfgang Girzalsky, and Ralf Erdmann
2 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 Proteins
INTRODUCTION
Isolation and Analysis of Microtubules and Associated Proteins
Roger D. Sloboda
PROTOCOLS
1 Isolation of Microtubules by Assembly/Disassembly Methods
Roger D. Sloboda
2 Isolation of Microtubules and Microtubule-Associated Proteins Using Paclitaxel
Roger D. Sloboda
3 Separation of Tubulin and Microtubule-Associated Proteins by Ion Exchange Chromatography
Roger D. Sloboda
4 Isolation of Microtubule-Based Motor Proteins by ATP Release from Paxlitaxel-Stabilized Microtubules
Roger D. Sloboda
5 Observation of Microtubule-Based Motor Protein Activity
Roger D. Sloboda
 
CHAPTER 12 Golgi Isolation
PROTOCOL
Golgi Isolation
Danming Tang and Yanzhuang Wang
 
CHAPTER 13 Isolation of Mitochondria from Cells and Tissues
INTRODUCTION
Isolation of Mitochondria from Cells and Tissues
David A. Clayton and Gerald S. Shadel
PROTOCOLS
1 Isolation of Mitochondria from Tissue Culture Cells
David A. Clayton and Gerald S. Shadel
2 Isolation of Mitochondria from Animal Tissue
David A. Clayton and Gerald S. Shadel
3 Purification of Mitochondria by Sucrose Step Density Gradient Centrifugation
David A. Clayton and Gerald S. Shadel
 
CHAPTER 14 Isolation of Chloroplasts from Plant Protoplasts
PROTOCOL
1 Isolation of Chloroplasts from Plant Protoplasts
Shiu-Cheung Lung, Matthew D. Smith, and Simon D.X. Chuong
 
CHAPTER 15 Subcellular Fractionation of Rough Microsomes
INTRODUCTION
Subcellular Fractionation of Rough Microsomes
David D. Sabatini
PROTOCOLS
1 Preparation of Rough Microsomes from Rat Liver
David D. Sabatini
2 Preparation of Crude Rough Microsomes from Dog Pancreas
David D. Sabatini
3 Preparation of Crude Rough Microsomes from Tissue Culture Cells
David D. Sabatini
 
CHAPTER 16 Purification of Ribosomes, Ribosomal Subunits, and Polysomes
INTRODUCTION
Purification of Ribosomes, Ribosomal Subunits, and Polysomes
Maria C. Rivera, Bruce Maguire, and James A. Lake
PROTOCOLS
1 Isolation of Ribosomes and Polysomes
Maria C. Rivera, Bruce Maguire, and James A. Lake
2 Isolation of Ribosomes by Chromatography
Bruce A. Maguire
3 Purification of 70S Ribosomes
Maria C. Rivera, Bruce Maguire, and James A. Lake
4 Purification of Polysomes
Maria C. Rivera, Bruce Maguire, and James A. Lake
5 Dissociation of Ribosomes into Large and Small Subunits
Maria C. Rivera, Bruce Maguire, and James A. Lake
 
CHAPTER 17 Isolation of Nuclei
INTRODUCTION
Isolation of Nuclei
Arash Nabbi and Karl Riabowol
PROTOCOLS
1 Rapid Isolation of Nuclei from Cells In Vitro
Arash Nabbi and Karl Riabowol
2 Isolation of Pure Nuclei Using a Sucrose Cushion
Arash Nabbi and Karl Riabowol
 
CHAPTER 18 The Nuclear Matrix: Fractionation Techniques and Analysis
INTRODUCTION
The Nuclear Matrix: Fractionation Techniques and Analysis
Rosemary H.C. Wilson, Emma L. Hesketh, and Dawn Coverley
PROTOCOL
1 Preparation of the Nuclear Matrix for Parallel Microscopy and Biochemical Analyses
Rosemary H.C. Wilson, Emma L. Hesketh, and Dawn Coverley
 
APPENDIX General Safety and Hazardous Material Information
 
Index