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Isolation of smooth‐type lipopolysaccharides to electrophoretic homogeneity
Author(s) -
Pupo Elder,
Hardy Eugenio
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600744
Subject(s) - chromatography , chemistry , limulus amebocyte lysate , molecular mass , elution , electrophoresis , lysis , homogeneous , escherichia coli , biochemistry , lipopolysaccharide , biology , enzyme , physics , gene , endocrinology , thermodynamics
The high structural heterogeneity of smooth‐type lipopolysaccharides (LPS) enormously complicates the isolation of their constituent molecular species. Proof of concept is given here on the feasibility of using preparative slab‐PAGE to isolate highly homogeneous smooth‐type LPS glycoforms. LPS species (from 3.6 to 14.2 kDa) from Escherichia coli K‐235 were separated by preparative slab‐PAGE and recovered by utilizing the combined on‐gel LPS reverse staining, extrusion, and passive elution techniques. As a result, 15 electrophoretically pure LPS fractions were obtained. T he LPS content in the recovered fractions ranged from 280 ng (intermediate mobility glycoforms) to 411 μg (highest mobility glycoforms). The quantities of LPS fractions were sufficient to allow quantitation of the Limulus amebocyte lysate (LAL) activities of these distinct‐molecular‐mass LPS species, in the range from (1.1 ± 0.1)×10 3 to (8.7 ± 0.3)×10 5 endotoxin units (EU)/mL, by standard LAL assay. We have thus definitively demonstrated that slab‐PAGE may be a suitable platform to more selectively purify individual glycoform fractions from smooth‐type LPS.