Subcellular Fractionation: A Laboratory Manual

Subject Area(s): Cell Biology; Biochemistry

By Paul Pryor, Hull York Medical School

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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
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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.

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