Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels.
A silver staining method used routinely for detecting bacterial lipopolysaccharide (LPS) in sodium dodecyl sulfate-polyacrylamide gels (C. Tsai and E. Frasch, Anal. Biochem. 119:115-119, 1982) appeared to be inappropriate for visualizing certain LPS preparations. It did not stain S-form fractions of polyagglutinable Pseudomonas aeruginosa LPS or several partly deacylated (alkali-treated) S-form LPS after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, these LPS preparations could be detected by anti-LPS sera after electroblotting onto nitrocellulose, thereby confirming their integrity and presence in the polyacrylamide gel. This is because LPS fractions containing a low number of fatty acids are washed out of the gel during the initial fixing step (40% ethanol-4% acetic acid, overnight). By omitting this fixing step, which was originally developed for detecting proteins, and by increasing the LPS oxidation time (from 5 to 20 min), we restored the ability to detect LPS fractions that otherwise would not be stained. These modifications did not affect the detection of other S- and R-form LPSs. Thus, differences in the number of fatty acids present in polyagglutinable P. aeruginosa LPS may result in a selective loss of fatty acid-deficient S-form LPS in these apparent R-form LPS preparations. This modified procedure provides a fast, simple, and sensitive way to analyze LPS in polyacrylamide gels despite the number of acyl groups present.
Fomsgaard
A
A
Max-Planck Institut für Immunbiologie, Freiburg, Federal Republic of Germany.
Freudenberg
M A
MA
Galanos
C
C
D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000737:chemistry; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000737:chemistry / Q000302:isolation & purification*; D011550:Pseudomonas aeruginosa / Q000032:analysis; D012834:Silver; D013194:Staining and Labeling / Q000379:methods*
D005227:Fatty Acids; D008070:Lipopolysaccharides; D012834:Silver