Rapid purification of extracted bacterial lipopolysaccharides by continuous free-flow electrophoresis.
The use of continuous free-flow electrophoresis for the purification of extracted lipopolysaccharides ( LPSs ) was investigated. Commercial (nucleic acid contaminated) LPS preparations, isolated by the hot phenol-water method of Westphal from Salmonella typhimurium and Escherichia coli 0111: B4, were analyzed. Continuous free-flow electrophoresis for purification of crude LPSs proved to be a rapid and useful means for the continuous purification of large amounts of LPS (more than 45 mg crude LPS per hr) and it showed good reproducibility and pure LPS. The electrophoretic profile of both crude LPSs obtained by continuous free-flow electrophoresis showed two distinct, sharp peaks; one representing the nucleic acid fraction and the other the LPS fraction. Under the continuous free-flow electrophoresis conditions employed, nucleic acid in the crude LPSs possessed low electrophoretic mobility, whereas LPS migration was negligible. Thus for both preparations pure LPS (no detectable nucleic acid) was obtained. Electrophoretic profiles of these purified LPSs on sodium dodecylsulfate-polyacrylamide gel electrophoresis were similar in both cases to those of crude LPS and of LPS purified by repeated ultracentrifugation. By immunological analysis using double immunodiffusion and immunoelectrophoresis, it was found that two components of crude E. coli 0111: B4 LPS were eliminated by continuous free-flow electrophoresis, but each component of purified E. coli 0111: B4 LPS was immunologically identical to the corresponding component in its crude LPS. In S. typhimurium LPS, none of its components were influenced by continuous free-flow electrophoresis but not by ultracentrifugation. In spite of these results, both purified LPSs possessed stronger mitogenic activity than each crude LPS. These results indicated that continuous free-flow electrophoresis is a useful means of purifying extracted crude LPS.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D008070:Lipopolysaccharides / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D012486:Salmonella typhimurium; D014461:Ultracentrifugation
D008070:Lipopolysaccharides