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. 2004 Apr;186(8):2253–2265. doi: 10.1128/JB.186.8.2253-2265.2004

FIG. 10.

FIG. 10.

Analysis of the wild-type (WT) and wbpB mutant flagellins by SDS-PAGE and Western immunoblotting. (A and B) Optimization of the sample preparation method showing enrichment of the samples in the flagellin band (band C) in glycine extracts (G) compared to soluble extracts (S); Ponceau red staining (A) and Western immunoblotting with anti-FlaA serum (B) after affinity purification and adsorption against an H. pylori flagellin A (flaA−) knockout mutant were used. (C and D) Enzymatic deglycosylation of flagellum preparations obtained by glycine extraction. + and − indicate the presence and absence of enzymatic deglycosylation, respectively. Coomassie staining (C) and Western immunoblotting with anti-FlaA antibody adsorbed against an E. coli lysate (D) were used. C. jejuni (CJ, strain 81-176) flagellum preparations obtained under the same conditions and bovine fetuine (BF) were used as positive controls for deglycosylation. Note that the anti-FlaA antibody readily detects C. jejuni glycosylated flagellins. All gels contained 10% acrylamide, and overexpressed and purified flagellins (FlaA and FlaB) were used as positive controls independently or as a mixture (A+B). MW, molecular mass standards.