Detection of lipopolysaccharides in polyacrylamide gels by transfer to nitrocellulose followed by immunoautoradiography with antibody and 125I-protein A: “LPS blotting”

Bradbury, W C; Mills, Scott D.; Preston, M A; Barton, L J; Penner, J L

doi:10.1016/0003-2697(84)90358-0

Cited by 16 publications

(3 citation statements)

References 13 publications

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“…The exact cause for this doublet pattern is unknown, but suggested explanations include storage artifact (Goldman and Leive 1980) and differences in growth medium composition (Palva and Makela 1980). Bradbury et al (1984) described a method for transferring electrophoretically resolved LPS to nitrocellulose and then immunologically detecting the bound LPS with antibodies. Immunoblotting of SDSMPAGE-resolved E. ictaluri LPS with hyperimmune anli-E. ictaluri AL83189 serum showed the ladderlike pattern of the LPS more clearly than silver staining.…”

Section: Discussionmentioning

confidence: 99%

Electrophoretic and Immunochemical Characterization of Lipopolysaccharide ofEdwardsiella ictalurifrom Channel Catfish

Newton

Triche

1993

Journal of Aquatic Animal Health

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Lipopolysaccharide (LPS) was purified from 40 isolates of Edwardsiella ictaluri by two methods: (1) enzyme digestion and hot aqueous phenol extraction and (2) enzyme digestion and gel exclusion chromatography. Purified LPS was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblot analysis. Both methods of purification yielded smooth LPS as evidenced by a ladderlike pattern of more than 40 LPS bands. With silver staining, both low-and high-molecular-mass LPS bands were seen. Lower-molecularmass LPS stained more intensely than higher-molecular-mass LPS bands, indicating a preponderance of lower-molecular-mass LPSs. Lipopolysaccharide bands from the 40 isolates migrated similarly within SDS-PAGE gels, indicating a high degree of structural similarity among the isolates examined. The ladderlike array was more easily seen with immunoblot analysis than with silver staining of SDS-PAGE gels. Additionally, immunoblot analysis revealed a high degree of antigenic similarity among the 40 isolates.

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Section: Discussionmentioning

confidence: 99%

Electrophoretic and Immunochemical Characterization of Lipopolysaccharide ofEdwardsiella ictalurifrom Channel Catfish

Newton

Triche

1993

Journal of Aquatic Animal Health

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“…The membranes were rinsed briefly in distilled water and washed twice with 100 ml of TBS containing 0.05% Tween 20. The membranes were then transferred to another dish containing a 1:3,000 dilution 1 and incubated for 1 h. The membranes were washed twice more with 100 ml of 0.05% Tween in TBS. The bands were developed by immersing the membranes for 30 min in a solution containing 0.05% horseradish peroxidase color development reagent (Bio-Rad Laboratories), 0.015% H202, and 16.7% methanol in TBS.…”

Section: Methodsmentioning

confidence: 99%

Electrophoretic and serological characterization of the lipopolysaccharides of Legionella pneumophila

Nolte¹,

Conlin²,

Motley³

1986

Infect Immun

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Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteinase-K-digested cell lysates to provide preliminary data on the LPS chemotypes for 20 strains of LegioneUa pneumophila (serogroups 1 to 8). The profiles of all strains except Chicago 2 (serogroup 6) were similar in the number, spacing, and size distribution of the bands visualized on silver-stained gels and were indicative of smooth LPS. However, compared with the bands from Salmonella minnesota smooth LPS, their banding pattern was much tighter, with three to four legionella bands for every salmonella band. The proteinase K digest of Chicago 2 was unique in that only two widely separated silver-stained bands were seen. LPS profiles of 10 serogroup 1 strains were identical, and the profile of Knoxville 1 was not altered by extensive in vitro passage. We used immunoblotting to investigate the serological specificities of the LPSs. When a rabbit antiserum prepared against a serogroup 1 strain was used to probe nitrocellulose sheets that bound LPS from strains belonging to eight different serogroups, it recognized only the LPS from the homologous serogroup. Similar results were observed with serogroup 2, 4, and 6 antisera. Our data indicate that L. pneumophila has a smooth-type LPS with an unusual banding pattern and that it is a serogroup-specific antigen.

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“…LPS was visualized on the gels by the silver staining procedure of Tsai and Frasch (32). LPS separated by gel electrophoresis was then transferred to nitrocellulose paper, as described by Bradbury et al (3). The blot was incubated sequentially with a 1 in 1,000 dilution of polyclonal O157-specific rabbit antiserum (Difco) and peroxidase conjugated to anti-rabbit immunoglobulin.…”

Section: Methodsmentioning

confidence: 99%

Polysaccharide side chains are not required for attaching and effacing adhesion of Escherichia coli O157:H7

et al. 1996

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Escherichia coli of the serotype O157:H7 is an enterohemorrhagic human pathogen which demonstrates attaching and effacing adhesion to colonocytes in vivo and to epithelial cells grown in tissue culture. Transposon TnphoA mutants of E. coli O157:H7 strain CL-8 were produced. Two of 300 alkaline phosphatase positive mutants, designated JB6 and JB27, did not express O157 side chains as assessed by agglutination with specific polyclonal O157 antiserum, silver staining of lipopolysaccharide extracts separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and Western immunoblots with polyclonal O157-specific antiserum. Both O157-negative mutants and the parent strain demonstrated localized adherence to HEp-2 cells when examined by Giemsa staining and bright-field microscopy. Furthermore, both O157-negative mutants showed enhanced adherence to HEp-2 cells compared with the parent strain when assessed by quantification of adherent bacterial CFUs. The parent strain, CL-8, and both of the mutants produced fluorescent foci when adherent bacteria and HEp-2 cells were stained with fluorescein isothiocyanate-labelled phalloidin. Transmission electron microscopy confirmed attaching and effacing adherence of strain CL-8 and the O157-negative mutants to HEp-2 cells. These findings indicate that mutants deficient in O157 polysaccharide repeats exhibit adherence to tissue culture cells in vitro and that O157 polysaccharide repeats are not required to produce the attaching and effacing lesion.

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Detection of lipopolysaccharides in polyacrylamide gels by transfer to nitrocellulose followed by immunoautoradiography with antibody and 125I-protein A: “LPS blotting”

Cited by 16 publications

References 13 publications

Electrophoretic and Immunochemical Characterization of Lipopolysaccharide ofEdwardsiella ictalurifrom Channel Catfish

Electrophoretic and Immunochemical Characterization of Lipopolysaccharide ofEdwardsiella ictalurifrom Channel Catfish

Electrophoretic and serological characterization of the lipopolysaccharides of Legionella pneumophila

Polysaccharide side chains are not required for attaching and effacing adhesion of Escherichia coli O157:H7

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