Summary
Producing high quality two-dimensional electrophoretic gels of bacterial cell membrane proteins is challenging because of non-membrane protein impurities within cell membrane preparations and the intractability of membrane proteins for solubilisation. Incubation of cell membrane preparations with sodium carbonate solution enriches for cell membrane proteins by stripping away other loosely associated protein contaminants from membranes (e.g., ribosomes, elongation factors). With the aid of strong sample solubilising reagents compatible with two-dimensional electrophoresis (2-DE), many of the carbonate-enriched membrane proteins can be separated using standard 2-DE techniques.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Molloy, M. P., Herbert, B. R., Slade, M. B., Rabilloud, T., Nouwens, A. S., Williams, K. L., Gooley, A. A. (2000) Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267, 2871–2871.
Fujiki, Y., Hubbard, A. L., Fowler, S., Lazarow, P. B. (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell. Biol.. 93, 97–102.
Molloy, M. P., Phadke, N. D., Maddock, J. R., Andrews, P. C. (2001) Two-dimensional electrophoresis and peptide mass-fingerprinting of bacterial outer membrane proteins. Electrophoresis 22, 1686–1696.
Nouwens, A. S., Cordwell, S. J. Larsen, M. R., Molloy, M. P., Gillings, M., Willicox, M. D. P., Walsh, B. J. (2000) Complementing genomics with proteomics: the membrane sub-proteome of Pseudomonas aeruginosa PA01. Electrophoresis 21, 3797–3809.
Sanchez, J-C., Hochstrasser, D., Rabilloud, T. (1999) In-gel sample rehydration of immobilized pH gradient, in Methods in molecular biology, vol. 112: 2D Proteome Analysis Protocols (Link, A.J., ed.), Humana Press, Totowa, NJ. pp. 221–225.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Molloy, M.P. (2008). Isolation of Bacterial Cell Membranes Proteins Using Carbonate Extraction. 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_30
Download citation
DOI: https://doi.org/10.1007/978-1-60327-064-9_30
Publisher Name: Humana Press
Print ISBN: 978-1-58829-722-8
Online ISBN: 978-1-60327-064-9
eBook Packages: Springer Protocols