Aberrant Migration of Lipopolysaccharide in Sodium Dodecyl Sulfate/Poyacrylamide Gel Electrophoresis

Abstract

Purified lipopolysaccharides of salmonellae strains were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Pre-electrophoresis of polyacrylamide gels had no apparent effect on one-dimensional silver-stained lipopolysaccharide profiles. However, without pre-electrophoresis, two-dimensional and three-dimensional patterns contained numerous bands with varied migration patterns compared to those in the one-dimension gels. The lipopolysaccharide was altered within the polyacrylamide gel during electrophoresis. Pre-electrophoresis of gels eliminated aberrant migration patterns.Electrophoresis of lipopolysaccharides (LPS) in polyacrylamide gels in the presence of an anionic detergent, sodium dodecyl sulfate (SDS/PAGE), has proved to be a powerful tool for separating and characterizing LPS [I -81. Interpretation of LPS SDSjPAGE patterns has been facilitated by the use of radiolabeling techniques with LPS mutants such as incorporation of isotopes into the 0-side chain, the core or the lipid A components and subsequent analysis of the distribution of isotopes in LPS SDSjPAGE patterns [2,5,6].The presence of SDS is thought to result in a reversible dissociation of LPS into subunits [3,9]. SDS probably binds to the lipid A region, perhaps to cationic sites plus adjacent hydrophobic regions [9]. Where it has been examined, the molar ratio of SDS bound to LPS is approximately half that bound to protein (0.63 versus 1.4) [C,]. Nonetheless, LPS has been reported to behave similarly to proteins in SDS/ PAGE, its migration characteristics depending upon size, not charge [1,4-81. In order to examine the possible contribution of LPS aggregation to the migration characteristics of LPS in the SDS/PAGE technique, LPSs of varying chemical composition were subjected to SDSjPAGE in one-dimensional, twodimensional, and three-dimensional (1 D, 2D, 3 D) systems. The use of an extremely sensitive silver-staining technique permitted visualization of very small quantities of LPS in the polyacrylamide gels [2]. MATERIALS A N D METHODS Li~n~ol~saccharide.sLPS from Salmonella typhimurium LT2 smooth (S) chemotype was extracted by the Westphal procedure [lo]. The phenol/chloroform/petroleum ether method of Galanos [I 1 ] was used to extract core-defective LPSs from S. typhimuriurn G3O/C21 deep rough (Re) and Salmonella minnesota R60 (Ra) and R4 (Rdz) mutants. LPS preparations contained < 1 "/, Abbreviations. LPS, lipopolysaccharide: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; ID, 2D, and 3D, oncdimensional, two-dimensional, and three-dimensional. Sodium Dodecyl S u l f a t e / P o l~a c r~l a m i~e Gel ElectrophoresisPreparations were subjected to SDS/PAGE using the Laemmli buffer system [13]. The 4 % stacking gel and the 12.5 XI separating gel did not contain SDS. Electrophoresis was done at 35 mA constant current, using Tris/glycine buffer with 0.1 : . . < SDS (pH 8.3) for approximately 2.5 h. Pre-electrophoresis for a minimum of 30 min was done before adding samples in some experiments; in most...

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus

“…Even the roughtype (Ra) LPS of S. typhimurium TV-119 appeared to have a slower-moving band above the fastest-moving band. A similar banding pattern had been observed with the Ra LPS of Salmonella minnesota (20). The slower-moving band had been proved to be an aggregate of the LPS molecules in the fastest-moving band by extensive gel electrophoresis studies (20).…”

“…A similar banding pattern had been observed with the Ra LPS of Salmonella minnesota (20). The slower-moving band had been proved to be an aggregate of the LPS molecules in the fastest-moving band by extensive gel electrophoresis studies (20). Multiple (more than two) widely separated LPS bands on SDS-PAGE gel have been reported for other gram-negative bacteria (2, 9, 14, 29, 42).…”

Identification of Candidates for a Subunit Vaccine against Extraintestinal Pathogenic Escherichia coli

“…Lipopolysaccharide (LPS) was prepared from S26 or BL21 E. coli strains as previously described (31). Briefly, overnight colonies were washed in PBS and resuspended in a protein lysis buffer.…”

Killing of gram-negative bacteria by lactoferrin and lysozyme.

“…All samples were treated with proteinase K for 1 h at 60°C and boiled for 10 min. Discontinuous SDS-PAGE using the Laemmeli system was performed with 4.5% stacking and 15% resolving gels and analysis by silver stain (34 (18,(39)(40)(41). Bacteria were grown to stationary phase, the cells centrifuged and washed.…”