Separation of bacterial capsular and lipopolysaccharides by preparative electrophoresis

Abstract

Preparative gel electrophoresis was used to separate and purify extracellular, capsular and lipopolysaccharides (EPSs, CPSs, and LPSs, respectively) from crude bacterial extracts. The procedure effectively separates CPS from LPSs. In addition discreet size ranges of these various polysaccharides can be isolated. The 'rough' (R-type), 'smooth' (S-type), and 'semi-smooth' LPSs were separated from one another. In addition different size classes of 'semi-smooth', or S-type LPS, can be separated. This procedure was demonstrated for diverse bacterial species, including the soil bacteria Rhizobium fredii, and the enteric bacterial species, Salmonella enteritidis and Proteus mirabilis. In the latter case, it was also possible to separate capsular polysaccharide from its lipid-bound form.

Isolation of smooth‐type lipopolysaccharides to electrophoretic homogeneity

“…In fact, purified LPS fractions generally showed several LPS species upon re-electrophoresis [21,22]. As compared to preparative DOC-PAGE in the tube gel format, the present slab-PAGE-based isolation methodology has also the advantage that it provides a more precise detection and isolation of individual LPS species [23,24]. Furthermore, in contrast to the tube gel DOC-PAGE technique, electrophoresis-associated salts or detergents are washed out from the gel matrix before LPS elution, and the recovered LPS fractions are freed of these contaminants.…”

The Structure of the Colony Migration Factor from PathogenicProteus mirabilis

“…1A), a property characteristic of acidic polysaccharides (32). The low molecular weight fraction silver-stained after being fixed in the presence or absence of Alcian blue, a feature that is characteristic of LPS (32). Thus, the crude Cmf-CPS fraction contained a high molecular weight Cmf-CPS DOC and a low molecular weight LPS (a lipo-oligosaccharide, LOS DOC ).…”

“…The DOC-PAGE analysis of the fractions resulted in a fraction that silverstained only after fixing the gel in the presence of Alcian blue (Fig. 1A), a property characteristic of acidic polysaccharides (32). The low molecular weight fraction silver-stained after being fixed in the presence or absence of Alcian blue, a feature that is characteristic of LPS (32).…”

Harnessing metal ion affinity for the purification of plasmid DNA

“…Interestingly, despite the high ionic strength of cell lysis buffer comparable to Tris-NaCl buffer (∼ 70 ms/cm), both Cu 2+ and Zn 2+ interactions with endotoxin were enhanced in cell lysis buffer with ∼ 90% endotoxin precipitated at 6 nmol metal ion/EU. Endotoxin is reported to exist in aggregated form in solution (Kim et al, 1996). The presence of ∼ 0.3% (w/v) SDS in the cell lysis buffer is likely to disaggregate the entangled endotoxin molecules, rendering electron-rich phosphoric acid group of endotoxin more available to the free metal ions, and thereby resulting in more effective interaction of free metal ions with endotoxin.…”