Rapid preparation of affinity-purified lipopolysaccharide samples for electrophoretic analysis.

Lipopolysaccharides (LPS) are the major components of the outer membrane of gram-negative bacteria. These surface molecules are relevant both for the outer membrane stability and for the interaction of the bacteria with other organisms and with the environment (18). Extensive literature is available concerning LPS physiology in different symbiotic (parasitic) and pathogenic host-bacterial interaction systems (5,20). Physiological, biochemical and chemical approaches to study LPS functions commonly require LPS extraction and purification. In the literature, LPS have been prepared by a number of different cell disruption procedures. Protocols for LPS isolation include organic solvent extractions followed by exhaustive dialysis or evaporation steps, and their modifications (11,22). An alternative procedure was described by Leive (17), which involves washing the bacteria with EDTA. In this procedure, incubation of cells with aqueous EDTA releases a significant proportion of LPS (ca. 30% to 50% in E. coli and Salmonella) without bacterial lysis, thus avoiding drastic protein, DNA and RNA contamination. All these protocols have been widely used for LPS chemical analysis (5,18), for the investigation of in vitro LPS biological activities (20) and also for analysis of LPS micro-heterogeneity using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) (4). As a preliminary tool before chemical analysis, electrophoretic characterization of LPS can give valuable information concerning size distribution of different LPS molecular forms and general indications about structural changes in the LPS that produce modifications in the electrophoretic mobility (8). A quick method for LPS characterization by SDSPAGE have been described by Hitchcock and Brown (12) using a whole SDS bacterial lysate. Although this method avoids tedious LPS preparations, it includes a strong protease treatment in the presence of SDS to clean samples after bacterial lysis. Technical aspects associated with different protocols for LPS isolation and its further SDS-PAGE analysis are summarized in Table 1. In this report, we describe an alternative, practical and rapid method for