Advertisement

Isolation of Bacterial Cell Membranes Proteins Using Carbonate Extraction

  • Mark P Molloy
Part of the Methods in Molecular Biology™ book series (MIMB, volume 424)

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.

Key Words

Carbonate extraction hydrophobicity membrane proteins sample solubilisation two-dimensional gel electrophoresis 

References

  1. 1.
    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.CrossRefPubMedGoogle Scholar
  2. 2.
    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.CrossRefPubMedGoogle Scholar
  3. 3.
    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.CrossRefPubMedGoogle Scholar
  4. 4.
    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.CrossRefPubMedGoogle Scholar
  5. 5.
    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.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Mark P Molloy
    • 1
  1. 1.Australian Proteome Analysis Facility LtdSydneyAustralia

Personalised recommendations