Summary
Advanced prefractionation strategies, in combination with highly sensitive and accurate mass spectrometers provide powerful means to detect and analyze low abundant proteins on the subcellular and organelle-specific level. Among enrichment techniques, subcellular fractionation has become the most commonly used. Its application gives access to less complex subproteomes and organelle constituents, facilitating downstream analysis. Furthermore, subcellular fractionation allows the identification of proteins that shuttle between different subcellular compartments in a stimulus dependent manner. As a paradigm of subcellular organelle isolation, we describe here endosomal purification protocols, based on differential centrifugation in continuous and discontinuous sucrose gradients. Described methods can be easily modified to isolate other organelles and are compatible with subsequent organelle- and functional organelle proteome analyses by, e.g., two-dimensional gel electrophoresis.
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Acknowledgments
Work in the Huber laboratory is supported by the Austrian Proteomics Platform (APP) within the Austrian Genome Program (GEN-AU), Vienna, Austria and by the Special research Program “Cell Proliferation and Cell Death in Tumors” (SFB021, Austrian Science Fund).
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Araùjo, M., Hube, L.A., Stasyk, T. (2008). Isolation of Endocitic Organelles by Density Gradient Centrifugation. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 424. Humana Press. https://doi.org/10.1007/978-1-60327-064-9_25
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DOI: https://doi.org/10.1007/978-1-60327-064-9_25
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