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Volume 136, Issue 8

Extraction of membrane antigens from and an assessment their serological activity by immunoblotting Free

Abstract

The efficacy and selectivity of chaotropic and phase-partitioning procedures for the extraction of membrane proteins from were compared with a standard Sarkosyl method. Major group 1, 2 and 3 outermembrane proteins (OMPs) of stained by Coomassie blue in SDS-PAGE gels had, respectively, apparent molecular masses of 81/82 kDa, 39–41 kDa and 30–32 kDa. The presence of these bands in the Sarkosyl extract of total membrane vesicles (TMVs) indicate that the procedure failed to selectively solubilize only inner-membrane proteins (IMPs). SDS-PAGE analyses also revealed the presence of OMPs and other additional bands following extraction of TMVs by butanol phase-partitioning or with extraction solutions based on the chaotropic reagents potassium thiocyanate (KSCN), sodium salicylate (SSC) and lithium acetate (LAE). OMPs are therefore not selectively extracted by any one of these procedures. Based on the number and staining intensity of extracted membrane-associated polypeptides, the efficacy of different extraction procedures could be graded in decreasing order as follows: KSCN, SSC, butanol and LAE. Both butanol and SSC were particularly effective in extracting group 3 OMPs. Sera from chronic excretor rams were used to identify zones of seroreactivity in immunoblots. Essentially, two reactivity patterns were seen: strong antibody binding against polypeptides in zones A (46-85 kDa), C (28–32 kDa) and D (18–22 kDa) in one, and additional reactivity against zones B (34–44 kDa) and E (13–18 kDa) polypeptides in the other. Irrespective of the method of membrane extraction, immunoblots using pooled ram sera collected from flocks with ovine brucellosis, and grouped on the basis of increasing complement fixation titre, displayed more intense reactivity against an increasing number of polypeptide bands. Membrane antigens extracted with KSCN and Sarkosyl possessed strong immunoblot reactivity in all five zones while OMPs generally showed the weakest reactivity. In contrast, immunoblot activity of polypeptides in SSC, butanol and LAE extracts were clustered in zones A, C, D and E. It is concluded that chaotropic and organic phase-partitioning procedures do not selectively extract OMPs from but represent simple and efficacious methods for generating antigen preparations enriched in membrane polypeptides.

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© Society For General Microbiology 1990
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1990-08-01
2024-04-25
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Extraction of membrane antigens from Brucella ovis and an assessment their serological activity by immunoblotting
Microbiology 136, 1615 (1990); https://doi.org/10.1099/00221287-136-8-1615
/content/journal/micro/10.1099/00221287-136-8-1615
/content/journal/micro/10.1099/00221287-136-8-1615
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