Rabbit anti serotype 4 capsule antibody

To verify glycoconjugate production and to select AKTA fractions for pooling, samples were subject to SDS-PAGE followed by coomassie staining or immunoblot. Rabbit anti-serotype 4 capsule antibody from the Statens Serum Institut, (SSI) Denmark was used at a dilution of 1:1000 and mouse anti-His monoclonal antibody (Abcam, UK) was used at a dilution of 1:10,000 to detect recombinant serotype 4 capsule and His-tagged AcrA respectively. HR6 antiserum was used to detect the Campylobacter heptasaccharide (S. Amber and M. Aebi, unpublished data). Secondary goat anti-rabbit IgG IRDye 800 and goat anti-mouse IgG IRDye 680 conjugates were used at a dilution of 1: 10 000. Fluorescent signal was detected using an Odyssey LI-COR detection system (LI-COR Biosciences UK Ltd.).

To verify glycoconjugate production, periplasmic extracts were analysed by SDS-PAGE and western blotting. Equal volumes of samples, previously normalised by optical density, were used. This enabled semi-quantitative comparison between samples on one blot. Samples were initially prepared with LDS sample buffer and separated on 4–12% bis–tris gels in MOPS buffer (Invitrogen, USA). The gels were then electroblotted onto a nitrocellulose membrane using a Trans-Blot Turbo Transfer System (Bio-Rad, USA). Rabbit anti-serotype 4 capsule antibody (Statens Serum Institut, Denmark) was used at a dilution of 1:1000 and mouse anti-His monoclonal antibody (Thermo Fisher Scientific, USA) was used at a dilution of 1:10,000 to detect recombinant serotype 4 capsule and His-tagged ExoA, respectively. Secondary goat anti-rabbit IgG IRDye 800 and goat anti-mouse IgG IRDye 680 conjugates (LI-COR Biosciences, USA) were used at a dilution of 1:10,000. Fluorescent signal in two channels, 700 and 800 nm, was detected with an Odyssey CLx LI-COR detection system, using a solid-state diode laser at wavelengths 685 and 785 nm, respectively (LI-COR Biosciences, USA). Subsequent semi-quantitative densitometry analysis of the glycoconjugates was performed using the Image Studio (LI-COR Biosciences, USA) analysis tool, as demonstrated in the supplementary material (Additional file 2: Figure S1).