The outer membrane of Gram-negative bacteria: lipid A isolation and characterization

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 966; p. 239
• Main Authors: Hankins, Jessica V, Madsen, James A, Needham, Brittany D, Brodbelt, Jennifer S, Trent, M Stephen
• Format: Journal Article
• Language: English
• Published: United States 2013
• Subjects: Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - isolation & purification; Carbohydrate Sequence; Chromatography, Thin Layer; Lipid A - chemistry; Lipid A - isolation & purification; Mass Spectrometry; Molecular Sequence Data
• ISSN: 1940-6029
• DOI: 10.1007/978-1-62703-245-2_15

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.