Summary
This chapter presents procedures for preparation and analysis of fractions enriched in plasma membranes from frozen tissue. It consists of a method for extraction and fractionation of membranes and a method for enzymatic digestion of membrane proteins without use of detergents. The method for isolation of membranes comprises a stepwise depletion of non-integral membrane proteins from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin and enrichment of the plasma membranes by a density gradient fractionation. Reduction, carboxymethylation, and digestion with endoproteinase Lys-C are carried out on non-solubilized membranes. The entire procedure allows processing and preparation of samples of minute amounts as 10–20 mg tissue and therefore can be extremely helpful for proteomic profiling of small pieces of tissue and clinical material.
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Acknowledgments
The author thanks Peter Aa. Nielsen (Proxeon A/S, Odense, Denmark) for excellent technical assistance and contribution to development of the described methods.
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Wiśniewski, J.R. (2008). Protocol to Enrich and Analyze Plasma Membrane Proteins from Frozen Tissues. In: Pflieger, D., Rossier, J. (eds) Organelle Proteomics. Methods in Molecular Biology™, vol 432. Humana Press. https://doi.org/10.1007/978-1-59745-028-7_12
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DOI: https://doi.org/10.1007/978-1-59745-028-7_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-779-2
Online ISBN: 978-1-59745-028-7
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