Expression of a recombinant 21 kDa polypeptide (rEhBvac21) from the EhV-ATPase B subunit and immunodetection of the endogenous protein by Western blot assays. rEhBvac21 polypeptide was IPTG induced in bacteria and purified through a Ni2+-NTA-agarose column as described in Methods. (a) Coomassie Blue stained 15% SDS-PAGE gel. Lane 1, protein molecular weight markers; lane 2, non-induced bacteria; lane 3, induced bacteria; lane 4, purified rEhBvac21 polypeptide. (b) Western blot assay of gel shown in (a), using anti-6His-tag monoclonal antibodies and horseradish peroxidase-conjugated anti-mouse IgG secondary antibodies (1:1,000), and revealed as described. (c) Western blot of purified rEhBvac21 polypeptide using: lane 1, mouse pre-immune serum or lane 2, mouse polyclonal anti-rEhBvac21 antibodies (1:1,000), and horseradish peroxidase-conjugated anti-mouse IgG secondary antibodies (1:1,000). (d) Ponceau stained nitrocellulose membrane of proteins (see Methods) separated by 10% SDS-PAGE. Lane 1, whole trophozoites; lane 2, soluble fraction; lane 3, EhkO-enriched fractions. (e) Immunodetection of proteins shown in (d), with mouse polyclonal anti-rEhBVac21 antibodies (1:20,000) and horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibodies (1:3,000) using the ECL Plus detection kit as described. Arrow shows the rEhBvac21 polypeptide. Arrow-head indicates the endogenous V-ATPse B subunit.