Immunoblot Analysis of L. pneumophila

L. pneumophila JR32 was lysed by sonication (Bandelin, Sonopuls, Berlin, Germany) in a lysis buffer (150 mM sodium chloride, 1% Triton X-100, 50 mM Tris; pH 8.0), followed by Proteinase K treatment (0.05 mg/mL). Afterwards, the samples were pipetted onto a nitrocellulose membrane (Macherey-Nagel, Düren, Germany), and an immunoblot test was performed as described by Raulf et al. [46 (link)]. Briefly, the membrane was blocked with a 5% milk solution and then incubated with the respective CLR-Fc fusion proteins (1 µg/mL). For detection, an HRP-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Labs, RRID: AB_2337586) was diluted 1:10.000 in TBS + 0.05% Tween-20. The signals were visualized using Amersham ECL Prime Western Blotting Detection Reagent (Thermo Fisher, MA, USA). Chemiluminescence was measured using a chemiluminescence imager (Bio-Rad Laboratories, Hercules, CA, USA).