Defining Membrane Protein Localization by Isopycnic Density Gradients

  • Rhys A. Dunstan
  • Iain D. Hay
  • Trevor LithgowEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)


In many bacteria, membrane proteins account for around one-third of the proteome and can represent much more than half of the mass of a membrane. Classic techniques in cell biology can be applied to characterise bacterial membranes and their membrane protein constituents. Here we describe a protocol for the purification of outer and inner membranes from Escherichia coli. The procedure can be applied with minor modifications to other bacterial species, including those carrying capsular polysaccharide attached to the outer membrane.

Key words

Sucrose density gradient Membrane biogenesis Beta-barrel proteins Cytoplasmic membrane Lipoproteins 


  1. 1.
    Dalbey RE, Wang P, Kuhn A (2011) Assembly of bacterial inner membrane proteins. Annu Rev Biochem 80:161–187CrossRefPubMedGoogle Scholar
  2. 2.
    Okuda S, Tokuda H (2011) Lipoprotein sorting in bacteria. Annu Rev Microbiol 65:239–259CrossRefPubMedGoogle Scholar
  3. 3.
    Kamio Y, Nikaido H (1976) Outer membrane of salmonella typhimurium: accessibility of phospholipid head groups to phospholipase c and cyanogen bromide activated dextran in the external medium. Biochemistry 15(12):2561–2570CrossRefPubMedGoogle Scholar
  4. 4.
    Smit J, Kamio Y, Nikaido H (1975) Outer membrane of salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J Bacteriol 124(2):942–958PubMedPubMedCentralGoogle Scholar
  5. 5.
    Osborn MJ, Gander JE, Parisi E, Carson J (1972) Mechanism of assembly of the outer membrane of salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem 247(12):3962–3972PubMedGoogle Scholar
  6. 6.
    Schnaitman CA (1970) Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli. J Bacteriol 104(2):890–901PubMedPubMedCentralGoogle Scholar
  7. 7.
    Yamato I, Anraku Y, Hirosawa K (1975) Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes. J Biochem 77(4):705–718CrossRefPubMedGoogle Scholar
  8. 8.
    Hobb RI, Fields JA, Burns CM, Thompson SA (2009) Evaluation of procedures for outer membrane isolation from Campylobacter jejuni. Microbiology 155(Pt 3):979–988CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Anwari K, Webb CT, Poggio S, Perry AJ, Belousoff M, Celik N, Ramm G, Lovering A, Sockett RE, Smit J, Jacobs-Wagner C, Lithgow T (2012) The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex. Mol Microbiol 84(5):832–844CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Selkrig J, Mosbahi K, Webb CT, Belousoff MJ, Perry AJ, Wells TJ, Morris F, Leyton DL, Totsika M, Phan MD, Celik N, Kelly M, Oates C, Hartland EL, Robins-Browne RM, Ramarathinam SH, Purcell AW, Schembri MA, Strugnell RA, Henderson IR, Walker D, Lithgow T (2012) Discovery of an archetypal protein transport system in bacterial outer membranes. Nat Struct Mol Biol 19(5):506–510CrossRefPubMedGoogle Scholar
  11. 11.
    Marchand CH, Salmeron C, Bou Raad R, Meniche X, Chami M, Masi M, Blanot D, Daffe M, Tropis M, Huc E, Le Marechal P, Decottignies P, Bayan N (2012) Biochemical disclosure of the mycolate outer membrane of Corynebacterium glutamicum. J Bacteriol 194(3):587–597CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Masson L, Holbein BE (1983) Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis. J Bacteriol 154(2):728–736PubMedPubMedCentralGoogle Scholar
  13. 13.
    Siegmund-Schultze N, Kroll HP, Martin HH, Nixdorff K (1991) Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness. J Gen Microbiol 137(12):2753–2759CrossRefPubMedGoogle Scholar
  14. 14.
    Jagannadham MV, Abou-Eladab EF, Kulkarni HM (2011) Identification of outer membrane proteins from an Antarctic bacterium Pseudomonas syringae Lz4W. Mol Cell Proteomics 10(6):M110 004549CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Bishop RE, Gibbons HS, Guina T, Trent MS, Miller SI, Raetz CR (2000) Transfer of palmitate from phospholipids to lipid a in outer membranes of gram-negative bacteria. EMBO J 19(19):5071–5080CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Charlson ES, Werner JN, Misra R (2006) Differential effects of yfgL mutation on Escherichia coli outer membrane proteins and lipopolysaccharide. J Bacteriol 188(20):7186–7194CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Steeghs L, de Cock H, Evers E, Zomer B, Tommassen J, van der Ley P (2001) Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant. EMBO J 20(24):6937–6945CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Dunstan RA, Hay ID, Wilksch JJ, Schittenhelm RB, Purcell AW, Clark J, Costin A, Ramm G, Strugnell RA, Lithgow T (2015) Assembly of the secretion pores GspD, Wza and CsgG into bacterial outer membranes does not require the Omp85 proteins BamA or TamA. Mol Microbiol 97(4):616–629CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Rhys A. Dunstan
    • 1
  • Iain D. Hay
    • 1
  • Trevor Lithgow
    • 1
    Email author
  1. 1.Department of Microbiology and Infection and Immunity Program, Biomedicine Discovery InstituteMonash UniversityMelbourneAustralia

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