Effect of pH and acrylamide concentration on the separation of lipopolysaccharides in polyacrylamide gels

Abstract

The technique of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the O-antigen of three strains ofPseudomonas aeruginosa, two strains, ofSalmonella typhimurium, and one strain ofEscherichia coli. No significant difference in separation and migration rate of sample was seen at the various acrylamide gel concentrations used. However, samples electrophoresed through acrylamide running gels at pH 6.8 migrated faster and the resolution of the high-molecular-weight O-antigen bands was greater than of the samples separated in gels at pH 8.8. On the basis of our observations, we could conclude that separation of the heterogeneous O-antigen in SDS-PAGE is probably due to differences in their charge densities and their molecular sizes. Also, pH 6.8 resolving gels are especially useful in the separation of high-molecular-weight O-antigen for epitope mapping by reaction with monoclonal antibodies in Western immunoblotting.

Literature Cited

  1. 1.

    Ciesielski CA, Blasser MJ, Wang W-LL (1986) Serogroup specificity ofLegionella pneumophila is related to lipopolysaccharide characteristics. Infect Immun 51:397–404

    Google Scholar 

  2. 2.

    Darveau RP, Hancock REW (1983) Procedure for isolation of bacterial lipopolysaccharides from both smooth and roughPseudomonas aeruginosa andSalmonella typhimurium strains. J Bacterial 155:831–838

    Google Scholar 

  3. 3.

    Hames BD, Rickwood D (1981) Gel electrophoresis of proteins. Oxford: IRL Press, pp 1–91

    Google Scholar 

  4. 4.

    Hitchcock PJ, Brown TM (1983) Morphological heterogeneity amongSalmonella lipopolysaccharide in silver-stained polyacrylamide gels. J Bacteriol 154:269–277

    Google Scholar 

  5. 5.

    Jann B, Reske K, Jann K (1975) Heterogeneity of lipopolysaccharides. Analysis of polysaccharide chain lengths by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Eur J Biochem 60:239–246

    Google Scholar 

  6. 6.

    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature 227:680–685

    Google Scholar 

  7. 7.

    Lam JS, MacDonald LA, Lam MYC, Duchesne LGM, Southam GG (1987) Production and characterization of monoclonal antibodies against serotype strains ofPseudomonas aeruginosa. Infect Immun 55:1051–1057

    Google Scholar 

  8. 8.

    MacIntyre S, Lucken R, and Owen P (1986) Smooth lipopolysaccharide is the major protective antigen for mice in the surface extract from IATS serotype 6 contributing to the polyvalentPseudomonas aeruginosa vaccine PEV. Infect Immun 52:76–84

    Google Scholar 

  9. 9.

    Mutharia LM, Hancock REW (1983) Surface localization ofPseudomonas aeruginosa outer membrane protein F using monoclonal antibodies. Infect Immun 42:1027–1033

    Google Scholar 

  10. 10.

    Perez-Perez GI, Blaser MJ, Bryner JH (1986) Lipopolysaccharide structures ofCampylobacter fetus are related to heat-stable serogroups. Infect Immun 51:209–212

    Google Scholar 

  11. 11.

    Pluschke G, Moll A, Kusecek B, Achtman M (1986) Sodium dodecyl sulfate-polyacrylamide, gel electrophoresis and monoclonal antibodies as tools for the subgrouping ofEscherichia coli lipopolysaccharide O18 and O23 antigens. Infect Immun 51:286–293

    Google Scholar 

  12. 12.

    Shapiro AL, Vinuela E, Maizel JV (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun 28:815–820

    Google Scholar 

  13. 13.

    Towbin M, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    Google Scholar 

  14. 14.

    Tsai C-M, Frasch CE (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115–119

    Google Scholar 

  15. 15.

    Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412

    Google Scholar 

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Correspondence to Dr. Joseph S. Lam.

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Duchesne, L.G.M., Lam, J.S., MacDonald, L.A. et al. Effect of pH and acrylamide concentration on the separation of lipopolysaccharides in polyacrylamide gels. Current Microbiology 16, 191–194 (1988). https://doi.org/10.1007/BF01568528

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Keywords

  • Migration
  • Electrophoresis
  • Polyacrylamide
  • Charge Density
  • Dodecyl