Chapter 5. Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling
- J. Robin Harris1,
- John Graham2,
- David Rickwood3
Published Online: 3 JUL 2006
DOI: 10.1002/0470033487.ch5
Copyright © 2006 John Wiley & Sons, Ltd
Book Title
Cell Biology Protocols
Additional Information
How to Cite
Graham, J. (2006) Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling, in Cell Biology Protocols (eds J. R. Harris, J. Graham and D. Rickwood), John Wiley & Sons, Ltd, Chichester, UK. doi: 10.1002/0470033487.ch5
Editor Information
- 1
Institute of Zoology, Johannes Gutenberg-Universität, University of Mainz, D-55099 Mainz, Germany
- 2
JG Research Consultancy, 34 Meadway, Upton Wirral CH49 6IQ, UK
- 3
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK
Publication History
- Published Online: 3 JUL 2006
- Published Print: 27 JAN 2006
ISBN Information
Print ISBN: 9780470847589
Online ISBN: 9780470033487
- Summary
- Chapter
Keywords:
- ER-Golgi Intermediate Compartment (ERGIC);
- plasma membrane domains;
- Laemmli gel electrophoresis system;
- cell monolayer culture;
- ball-bearing homogenizer;
- semi-dry blotting;
- enhanced chemiluminescence (ECL);
- Beckman Fraction Recovery System;
- Labconco Auto Densi-flow
Summary
This chapter contains sections titled:
Introduction
Methods available
Plasma membrane domains
Analysis of membrane compartments in the endoplasmic reticulum–Golgi–plasma membrane pathway
Separation of membrane vesicles from cytosolic proteins
Endocytosis
Protocols 5.1 Separation of basolateral and bile canalicular plasma membrane domains from mammalian liver in sucrose gradients
Protocols 5.2 Isolation of rat liver sinusoidal domain using antibody-bound beads
Protocols 5.3 Fractionation of apical and basolateral domains from Caco-2 cells in a sucrose gradient
Protocols 5.4 Fractionation of apical and basolateral domains from MDCK cells in an iodixanol gradient
Protocols 5.5 Isolation of lipid rafts
Protocols 5.6 Isolation of caveolae
Protocols 5.7 Analysis of Golgi and ER subfractions from cultured cells using discontinuous sucrose–D2O density gradients
Protocols 5.8 Analysis of Golgi, ER, ERGIC and other membrane compartments from cultured cells using continuous iodixanol density gradients
Protocols 5.9 Analysis of Golgi, ER, TGN and other membrane compartments in sedimentation velocity iodixanol density gradients (continuous or discontinuous)
Protocols 5.10 SDS–PAGE of membrane proteins
Protocols 5.11 Semi-dry blotting
Protocols 5.12 Detection of blotted proteins by enhanced chemiluminescence (ECL)
Protocols 5.13 Separation of membranes and cytosolic fractions from (a) mammalian cells and (b) bacteria
Protocols 5.14 Analysis of early and recycling endosomes in preformed iodixanol gradients; endocytosis of transferrin in transfected MDCK cells
Protocols 5.15 Analysis of clathrin-coated vesicle processing in self-generated iodixanol gradients; endocytosis of asialoglycoprotein by rat liver
Protocols 5.16 Polysucrose–Nycodenz® gradients for the analysis of dense endosome–lysosome events in mammalian liver
References