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

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.