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Raft-like membranes from the trans-Golgi network and endosomal compartments

Nature Protocols volume 8, pages 2429–2439 (2013)Cite this article

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Abstract

This article describes a procedure to prepare a raft-like intracellular membrane fraction enriched for the trans-Golgi network (TGN) and endosomal compartments. The initial step in this technique involves cell disruption by homogenization, followed by clearance of the plasma membrane, late endosomes, mitochondria and the endoplasmic reticulum by differential sedimentation. Carbonate treatment, sonication and sucrose density-gradient ultracentrifugation are subsequently used to isolate the target membranes. The isolated subcellular fraction contains less than 1% of the total cellular proteins, but it is highly enriched for syntaxin-6 and Rab11. Typically, 40–60% of the cellular pool of GM1 glycosphingolipid and 10–20% of the total cellular cholesterol cofractionate with this buoyant membrane fraction. Given the role of GM1 as a cell-surface receptor for the cholera toxin and that levels of both GM1 and cholesterol in the TGN-endosomal compartment are upregulated in some inherited diseases, this protocol can potentially be applied to the analysis of disease-associated changes to GM1-enriched intracellular membranes. The isolated membranes are very well separated from caveolin-rich domains of the plasma membrane, the TGN and recycling endosomes. The entire protocol can be completed in as little as 1 d.

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Figure 1
Figure 2: Typical distribution of cholesterol and GM1 ganglioside lipids in the sucrose density-gradient fractions 1–12 and the p8000 and p1000 pellet fractions.
Figure 3: Typical sucrose density profile of the sucrose step gradient (fractions 1–12), as determined by measurements of refractive index.
Figure 4: Western blots illustrating the typical distributions of proteins targeted to different subcellular membranes.

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Acknowledgements

Financial support from the Royal Free Charity is gratefully acknowledged. I thank J. Hsuan and M. Pinzani for the use of laboratory equipment and facilities, and K.M.E. Chu for assistance with Figure 1.

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  1. Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, UK

    Mark G Waugh

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M.G.W. designed and performed all experiments, analyzed the data and wrote the paper.

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Waugh, M. Raft-like membranes from the trans-Golgi network and endosomal compartments. Nat Protoc 8, 2429–2439 (2013). https://doi.org/10.1038/nprot.2013.148

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