The Outer Membrane of Gram-Negative Bacteria: Lipid A Isolation and Characterization

  • Jessica V. Hankins
  • James A. Madsen
  • Brittany D. Needham
  • Jennifer S. Brodbelt
  • M. Stephen Trent
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 966)

Abstract

The isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) are important methodologies utilized to gain understanding of the Gram-negative cell envelope. Here, we describe protocols often employed by our laboratory for small- and large-scale isolation of lipid A from bacterial cells. Additionally, we describe various methodologies including isolation of radiolabeled lipid A, thin layer chromatography, and various mass spectrometry methods. Tandem mass spectrometry is an integral tool for the structural characterization of lipid A molecules, and both coventional collision induced dissociation (CID) and new ultraviolet photodissociation (UVPD) methods are described.

Key words

Lipid A isolation Bligh–Dyer Thin layer chromatography Lipopolysaccharide Mass spectrometry Collision induced dissociation Photodissociation (PD) 
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Notes

Acknowledgments

This work was supported by National Institutes of Health (NIH) Grants AI064184 and AI76322 to M.S.T. and Welch Foundation Grant F1155 and NIH RO1 GM103655 to J.S.B.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Jessica V. Hankins
    • 1
  • James A. Madsen
    • 2
  • Brittany D. Needham
    • 3
  • Jennifer S. Brodbelt
    • 2
  • M. Stephen Trent
    • 1
    • 3
  1. 1.Section of Molecular Genetics and MicrobiologyThe University of Texas at AustinAustinUSA
  2. 2.Department of Chemistry and BiochemistryThe University of Texas at AustinAustinUSA
  3. 3.The Institute of Cellular and Molecular BiologyThe University of Texas at AustinAustinUSA

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