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. Subcellular Fractionation is therefore an essential laboratory resource for all cell biologists seeking a comprehensive collection of dependable subcellular fractionation methods.
1. Introduction: Subcellular Fractionation
—An introduction to cellular compartmentalisation and fractionation techniques. Discussion of fractionation of different samples (plant, human/animal including tissue culture cells), yeast and bacteria.
—Methods of fractionation (ways to break open/homogenise tissues) and then organelle/sample isolation such as differential centrifugation, density gradient centrifugation and direct isolation (magnetic, affinity chromatography).
2. Plasma membrane isolation using immobilised concanavalin A
3. Isolation of clathrin coated vesicles from tissue culture cells Stephen Royle
4. Purification of endosomes from cells
Lukas Huber,Mariana Eca, Guimaraes de Araujo, Giorgia Lamberti
5. The synaptosome as a model system for studying synaptic physiology
6. Isolation of phagosomes from tissue culture cells
7. Isolation of exosomes from cell culture media, blood plasma, serum, urine and cerebrospinal fluid
Emily Zeringer, Mu Li, Tim Barta, Alexander V. Vlassov
8. Isolation of lysosomes from rat tissue and tissue culture cells
9. Isolation of Specialised Secretory Compartments: GLUT4 Storage Vesicles
Jessica Sadler, Christopher Lamb, Gwyn Gould, Nia Bryant
10. Isolation of peroxisomes from yeast
Jana Cramer, Daniel Effelsberg, Wolfgang Girzalsky, Ralf Erdmann
11. Isolation of microtubules, MAPs, and mictrotubule-dependent motor proteins
12. Golgi isolation
Danming Tang, Yanzhuang Wang
13. Isolation of mitochondria from cells and tissues
D.A. Clayton, HHMI Janelia Farm with Gerald Shadel
14. Topic: Chloroplast isolation
15. Subcellular fractionation of rough microsomes
16. Purification of ribosomes, ribosomal subunits, and polysomes
M. Rivera, A.M. Aguinaldo, T. Atha, J. Lake, A. Scheinman
Isolation of Ribosomes by Chromatography
Purification of Ribosomes, Ribosomal Subunits, and Polysomes
Isolation of Ribosomes and Polysomes by Sucrose Density Gradient Centrifugation
Purification of 70S Ribosomes
Purification of Polysomes
Dissociation of Ribosomes into Large and Small Subunits
17. Rapid isolation of nuclei from cells in vitro
18. The nuclear matrix: Preparation for microscopy and biochemical analysis
Dawn Coverley, Rosemary Wilson
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