Analysis of lipopolysaccharides of Bacteroides fragilis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroblot transfer

Cousland, Gary

doi:10.1016/0378-1097(83)90116-7

Cited by 5 publications

(6 citation statements)

References 3 publications

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“…This was done by the method described by Cousland & Poxton (1983), with the Bio-Rad Immunoblot reagents, except that the nitrocellulose membrane was 0.2 pm pore size (Sartorius). Antigens were either electrophoretically transferred, or applied directly in 1 p1 volumes as dots for dot-blotting.…”

Section: Methodsmentioning

confidence: 99%

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Immunochemistry of the Surface Carbohydrate Antigens of Bacteroides fragilis and Definition of a Common Antigen

Poxton

Brown

1986

Microbiology

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The components extracted by aqueous phenol from whole cells of Bacteroides fragilis were analysed by SDS-PAGE and immunoblotting and shown to consist of a series of strain-specific, cross-reactive and common antigens, Regularly-spaced ladder patterns on silver-stained gels indicated that in most strains the LPS was present as a predominantly smooth type, but with chain lengths of varying molecular mass, ranging within each particular strain from essentially rough forms to long chain-length smooth forms. The rough form of the LPS at the gel front possessed an antigen common to most of the strains investigated. Another antigen, which migrated behind the rough LPS on SDS gels, was common to all strains of the species. The smooth LPS forms and the other high molecular mass components were strain-specific antigens. Previously published methods are not capable of producing pure LPS or capsular polysaccharide for this organism. claimed to have developed methods for the preparation of pure capsular polysaccharide free from LPS, and of LPS free from capsular polysaccharide, protein and nucleic acid. Starting with an aqueous phenol extract of whole cells, capsular polysaccharide was prepared by gel filtration in the presence of a detergent, whereas LPS was prepared by further extraction with a phenol/chloroform/petroleum spirit mixture. The authors suggested that the LPS of B.fragi1i.s is a rough-type molecule and is both chemically and antigenically similar in all the strains investigated.The development of a method for demonstrating the heterogeneity of LPS on polyacrylamide gels (Tsai & Frasch, 1982) has been applied to many species of Gram-negative bacteria. In a preliminary report (Cousland & Poxton, 1983), we showed that B. fragilis possesses a complex mixture of aqueous phenol-extractable surface carbohydrate antigens. A series of closely spaced bands in a ladder pattern on the silver-stained polyacrylamide gel was reminiscent of the pattern produced by the smooth form of LPS from other bacteria. This led us to suggest that B. fragilis possesses smooth type LPS. We also showed that a common antigen ran at the gel front and was associated in some unknown way with the rough form of the molecule.

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Section: Methodsmentioning

confidence: 99%

“…In a preliminary report (Cousland & Poxton, 1983), we showed that B. fragilis possesses a complex mixture of aqueous phenol-extractable surface carbohydrate antigens. A series of closely spaced bands in a ladder pattern on the silver-stained polyacrylamide gel was reminiscent of the pattern produced by the smooth form of LPS from other bacteria.…”

mentioning

confidence: 94%

Immunochemistry of the Surface Carbohydrate Antigens of Bacteroides fragilis and Definition of a Common Antigen

Poxton

Brown

1986

Microbiology

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“…This was essentially by the method of Towbin et al [19] with BioRad Immunoblot immunoassay, as described previously [20].…”

Section: Electroblot Transfer and Enzyme Immunoassaymentioning

confidence: 99%

The association on SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins ofPseudomonas aeruginosaas revealed by monoclonal antibodies and Western blotting

Poxton

Bell

Barclay

1985

FEMS Microbiology Letters

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Monoclonal antibodies raised against single serotype components of a Pseudomonas aeruginosa vaccine have been shown to bind to the O antigen region of lipopolysaccharide (LPS). Outer membrane (OM) proteins, prepared by detergent treatment of envelope fractions and by EDTA/sonication treatment of whole cells, were separated on sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), electrophoretically transferred to nitrocellulose membrane and reacted with LPS‐specific monoclonal antibodies. The patterns produced revealed that many of the protein bands were in fact protein‐LPS complexes.

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“…This was based on the method of Towbin et al (1979) as described by Cousland and Poxton (1983) with BioRad immunoblotting reagents, but the nitrocellulose membrane was of 0-2-pm pore size (Sartorius). For dot blotting, 2 pl of aqueous phenol-extracted LPS was applied directly to the nitrocellulose.…”

Section: Immunoblot Transfermentioning

confidence: 99%

Immunochemistry of the cell surfaces of Bacteroides bivius and Bacteroides disiens

Brown

Hornett

Poxton³

1989

Journal of Medical Microbiology

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Summary. Outer membranes were extracted from seven strains of Bacteroides bivius and six strains of B. disiens by the Sarkosyl method. Lipopolysaccharides (LPS) were extracted from the same strains by the Proteinase K method, and from three strains of each species by an aqueous phenol method. Analysis of the outer-membrane proteins by SDS-PAGE demonstrated that, within a species, very similar patterns with many shared or common bands were produced, but there were sufficient differences between species to allow separation. Immunoblotting with antisera raised against whole cells of each of the type strains showed that many antigens were shared between species. Smooth LPS was present in both species. By immunoblotting, the 0-antigen of B. disiens was shown to be common to all six strains, and there was no cross-reaction between the B. disiens antiserum and B. bivius LPS. The 0-antigen of B. bivius was not detected by immunoblotting with homologous antiserum, but antiserum to B. bivius reacted with a series of common low molecular mass antigens that were present in LPS preparations from strains of both species.

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Analysis of lipopolysaccharides of Bacteroides fragilis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroblot transfer

Cited by 5 publications

References 3 publications

Immunochemistry of the Surface Carbohydrate Antigens of Bacteroides fragilis and Definition of a Common Antigen

Immunochemistry of the Surface Carbohydrate Antigens of Bacteroides fragilis and Definition of a Common Antigen

The association on SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins ofPseudomonas aeruginosaas revealed by monoclonal antibodies and Western blotting

Immunochemistry of the cell surfaces of Bacteroides bivius and Bacteroides disiens

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