Subcellular Fractionation: A Laboratory Manual

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.

(preliminary)

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
Sue-Hwa Lin

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
Gareth Evans

6. Isolation of phagosomes from tissue culture cells
Paul Pryor

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
Paul Pryor

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
D.D. Sabatini

16. Purification of ribosomes, ribosomal subunits, and polysomes
M. Rivera, A.M. Aguinaldo, T. Atha, J. Lake, A. Scheinman

PROTOCOLS
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
Karl Riabowol

18. The nuclear matrix: Preparation for microscopy and biochemical analysis
Dawn Coverley, Rosemary Wilson

Glossary
Appendix
References
Index