Abstract
Polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate or sodium deoxycholate (SDS- or DOC-slab-PAGE) is a powerful technique for the separation of smooth(S)-type bacterial lipopolysaccharides (LPS). In order to recover the individual LPS species from the polyacrylamide gel for subsequent analyses, a sensitive, nondestructive reverse staining of slab-PAGE-separated LPS has been developed. The individual reverse-stained LPS bands can be rapidly and efficiently recovered into an aqueous 5% triethylamine solution when they are extruded to produce fine gel microparticles. Based on these principles, an isolation methodology that combines preparative slab-PAGE, reverse staining, extrusion, and passive elution can be used to isolate, to electrophoretic homogeneity, micrograms to hundreds of micrograms of individual LPS species successfully from smooth-type LPS mixtures.
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References
Tsai CM, Frasch CE (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115–119
Ohta M, Rothmann J, Kovats E, Pham PH, Nowotny A (1985) Biological activities of lipopolysaccharide fractionated by preparative acrylamide gel electrophoresis. Microbiol Immunol 29:1–12
Komuro T, Yomota C, Kimura T, Galanos C (1989) Comparison of R- and S-form lipopolysaccharides fractionated from Escherichia coli UKT-B lipopolysaccharide in pyrogen and Limulus tests. FEMS Microbiol Lett 51:79–83
Hardy E, Pupo E, Castellanos-Serra L, Reyes J, Fernandez-Patron C (1997) Sensitive reverse staining of bacterial lipopolysaccharides on polyacrylamide gels by using zinc and imidazole salts. Anal Biochem 244:28–32
Hardy E, Pupo E, Santana H, Guerra M, Castellanos-Serra LR (1998) Elution of lipopolysaccharides from polyacrylamide gels. Anal Biochem 259:162–165
Pupo E, Lopez CM, Alonso M, Hardy E (2000) High-efficiency passive elution of Âbacterial lipopolysaccharides from Âpolyacrylamide gels. Electrophoresis 21:526–530
Pupo E, Phillips NJ, Gibson BW, Apicella MA, Hardy E (2004) Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: high-resolution separation and molecular weight determination of lipooligosaccharides from Vibrio fischeri strain HMK. Electrophoresis 25:2156–2164
Gulin S, Pupo E, Schweda EK, Hardy E (2003) Linking mass spectrometry and slab-polyacrylamide gel electrophoresis by passive elution of lipopolysaccharides from reverse-stained gels: analysis of gel-purified lipopolysaccharides from Haemophilus influenzae strain Rd. Anal Chem 75:4918–4924
Pupo E, Hardy E (2007) Isolation of smooth-type lipopolysaccharides to electrophoretic homogeneity. Electrophoresis 28:2351–2357
Komuro T, Galanos C (1988) Analysis of Salmonella lipopolysaccharides by sodium deoxycholate-polyacrylamide gel electrophoresis. J Chromatogr 450:381–387
Acknowledgments
The author is a research fellow of the Alexander von Humboldt Foundation (Germany). The contribution of Prof. Dr. E. Hardy (University of Havana, Cuba) to the development of the methodology described here is specially acknowledged.
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Pupo, E. (2011). Isolation of Smooth-Type Lipopolysaccharides to Electrophoretic Homogeneity. In: Holst, O. (eds) Microbial Toxins. Methods in Molecular Biology, vol 739. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-102-4_9
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DOI: https://doi.org/10.1007/978-1-61779-102-4_9
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Publisher Name: Humana Press, Totowa, NJ
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