Comparison of two silver staining techniques for detecting lipopolysaccharides in polyacrylamide gels.

The classic silver staining method for detecting bacterial lipopolysaccharides (LPSs) in polyacrylamide gels (C. Tsai and C. E. Frasch, Anal. Biochem. 119:115-119, 1982) was at least 20 times more sensitive than the modified silver staining method (A. Fomsgaard, M. A. Freudenberg, and C. Galanos, J. Clin. Microbiol. 28:2627-2631, 1990) for detecting LPS from the bacterium Moraxella osloensis. However, the classic method is only about three to four times more sensitive than the modified method for detecting LPSs from Escherichia coli J5, EH100, and O111:B4 or Salmonella enterica serovar Typhimurium. The reduction of sensitivity is due to omission of the initial fixing step in the modified method. The retention of LPS fractions in the gels during fixing and/or oxidation may depend on the structures of their lipid A moieties. Lipopolysaccharides (LPSs) play a major role in the pathogenesis of gram-negative infections (1). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining has been used extensively to characterize LPS (3, 11, 12). Tsai and Frasch (11) first reported a highly sensitive classic silver staining method for detecting LPS in polyacrylamide gels. This classic method can detect even less than 5 ng of the rough type of LPS. However, Fomsgaard et al. (2) revealed that the classic method did not stain certain LPS preparations containing a low number of fatty acids, which were washed out of the gels during the initial fixing step (40% ethanol–5% acetic acid, overnight). Thus, they developed a modified silver staining method by omitting the fixing step and increasing the LPS periodic acid oxidation (the second step) time from 5 min to 20 min to restore the ability to detect all LPSs. We purified LPS from Moraxella osloensis, a bacterium associated with a slug-parasitic nematode, Phasmarhabditis hermaphrodita (8, 9). M. osloensis LPS is an active endotoxin against the slug Deroceras reticulatum (10). Analysis of different quantities of M. osloensis LPS or four commercially available LPSs from other bacteria by SDS-PAGE followed by each of the two methods revealed that the modified method was less sensitive than the classic method. M. osloensis LPS was purified by classical phenol-water extraction (13), with modification as described by Gu et al. (3), from 3-day pure cultures of M. osloensis supplied by MicroBio, Ltd., Cambridge, United Kingdom. LPS preparations from Escherichia coli J5, EH100, and O111:B4 strains and Salmonella enterica serovar Typhimurium were purchased from Sigma Chemical Company, St. Louis, Mo. LPS preparations were treated for 5 min at 100°C in 0.05 M Tris-HCl buffer (pH 6.8) containing 2% (wt/vol) SDS, 10% (wt/vol) sucrose, and 0.01% bromophenol blue. Ten microliters of each sample was then loaded on precast Ready Gel Tris-HCl polyacrylamide gels (86 by 68 by 1.0 mm) containing 4 and 15% acrylamide in the stacking and separating gels, respectively (Bio-Rad Laboratories, Inc., Hercules, Calif.). Electrophoresis was performed at 12 mA in the stacking gels and 25 mA in the separating gels until the bromophenol blue had run about 6.7 cm. LPSs in the gels were visualized by either the classic method (11) or the modified method (2). The sensitivities of the two methods were compared by using from 50 ng to 5 g of M. osloensis LPS (Fig. 1). The LPS was revealed to be a rough-type LPS, because only one main band was detected in the gels by both methods. The band patterns obtained at 50 ng by the classic method were equivalent to or better than those obtained at 1 g by the modified method. Therefore, the classic method is at least 20 times more sensitive than the modified method for detecting M. osloensis LPS. The sensitivities of the two methods were also compared by