Elution of lipopolysaccharides from polyacrylamide gels.
A simple procedure for elution in water of bacterial lipopolysaccharides (LPS) from sodium dodecyl sulfate-polyacrylamide gels is described. It consists of the combination of three principal steps: first, highly sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole (negative or reverse staining); second, washing of the individual LPS band in a solution of a zinc-complexing agent (e.g., 100 mM EDTA); and finally, elution of the LPS (100-200 microliters water for a 0.5-microgram LPS band) from gel microparticles for 3 h at room temperature. Using this procedure, we have successfully eluted a variety of LPS forms from Bordetella pertussis, Escherichia coli 0111:B4, E. coli K-235, Salmonella enteritidis, and Pseudomonas aeruginosa. Elution recovery of rough or semismooth LPS was about 70-80%, while that of smooth LPS was only about 10%. Eluted LPS was biologically active as tested by limulus amebocyte lysate and TNF-alpha assays.
Hardy
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E
Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Pupo
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E
Santana
H
H
Guerra
M
M
Castellanos-Serra
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LR
D000180:Acrylic Resins; D001886:Bordetella pertussis / Q000737:chemistry / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000302:isolation & purification* / Q000494:pharmacology; D008832:Microchemistry; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000276:immunology; D012477:Salmonella enteritidis / Q000737:chemistry / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000180:Acrylic Resins; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; C016680:polyacrylamide gels