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. 2019 May 10;9:7212. doi: 10.1038/s41598-019-43691-9

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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OMVs subjected to density gradient centrifugation exhibit high purity. (a) Transmission electron microscopy analysis of OMV preparations from wildtype V. cholerae V:5/04 (wt-OMV) and its VCC deletion mutant (Δvcc-OMV) before (wt-OMV-c and Δvcc-OMV-c) and after (wt-OMV-p and Δvcc-OMV-p) density gradient centrifugation. Arrows indicate flagellin filaments. Bars in the lower right of micrographs indicate 100 nm. (b) Nanoparticle tracking analysis of density gradient centrifugation purified OMVs from wildtype V. cholerae V:5/04 (wt-OMV-p) and its VCC deletion mutant (Δvcc-OMV-p). (c) Coomassie-brilliant-blue-stained SDS-PAGE gel and (d) immunoblot analysis of the same gel using polyclonal anti-VCC antiserum. Samples analysed: wt-OMV-c (lane 1), Δvcc-OMV-c (lane 2), wt-OMV-p (lane 3) and Δvcc-OMV-p (lane 4). Molecular weight markers were run in lane M. Molecular weights of the markers are given in kDa to the left of the gel in (c). Arrows in (d) indicate the position of the pro-VCC and the mature VCC protein, respectively. The full-length SDS-PAGE gel and its anti-VCC immunoblot are shown in Supplementary Fig. S1.