Mouse monoclonal anti e coli lps iggs clone 2d7 1

Following the purification of EVs, the proteins and associated lipopolysaccharides (LPS) were analyzed by dot blot on a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Eight µg (lipid amount) of each type of EVs were spotted. The membrane was blocked for 30 min in 5% non-fat dry milk in [50 mM Tris, 200 mM NaCl, pH 7.5, 0.1% Tween-20] (TBS-T), then incubated overnight in 2.5% non-fat dry milk in TBS-T containing either mouse monoclonal anti-E. coli LPS IgGs (clone 2D7/1, ab35654, Abcam, Cambridge, UK) diluted 1/2000 or rabbit anti-OmpA polyclonal antibodies (Epigentek, East Farmingdale, NY, USA) diluted 1/2000. After two washes of 10 min each in TBS-T, the membranes were incubated for 3 h, respectively in 2.5% non-fat dry milk in TBS-T containing anti-mouse polyclonal antibodies coupled to horse radish peroxidase [HRP] and diluted 1/10,000 or containing HRP-conjugated anti-rabbit IgG F(ab′)₂ fragment (Sigma) diluted 1/10,000. After washes in TBS-T, the membranes were revealed by chemiluminescence using a Clarity Western substrate kit (Bio-Rad) and photographed using a ChemiDoc apparatus (Bio-Rad).

OMV solutions corresponding to 7.5 µg of each type of OMV lipids were mixed with 4 µL of 4X Laemmli sample buffer (Bio-Rad). Whole cell extracts of each bacterial clone (corresponding to 3 µg of proteins) were mixed with 4 µL of 4X Laemmli sample buffer (Bio-Rad). Following SDS-PAGE, the proteins and associated LPS were transferred from the polyacrylamide gel to a nitrocellulose membrane (Bio-Rad) using a Trans-Blot turbo apparatus (Bio-Rad) according to the manufacturer’s recommendations. The membrane was blocked for 30 min in 5% non-fat dry milk in TBS-T [50 mM Tris, 200 mM NaCl, pH 7.5, 0.1% Tween-20], then incubated overnight in 2.5% non-fat dry milk in TBS-T containing either mouse monoclonal anti E. coli LPS IgGs (clone 2D7/1 (ab35654), Abcam, Cambridge, United Kingdom) diluted 1/2000 or rabbit anti-OmpA polyclonal antibodies (Epigentek, Farmingdale, United States) diluted 1/2000. After two washes of 10 min each in TBS-T, the membranes were incubated for 3 h, respectively, in 2.5% non-fat dry milk in TBS-T containing anti-mouse polyclonal antibodies coupled to horse radish peroxidase [HRP] and diluted 1/10,000 or containing HRP-conjugated anti-rabbit IgG F(ab’)₂ fragment (Sigma) diluted 1/10,000. After washes in TBS-T, the membranes were revealed by ECL using a Clarity Western substrate kit (Bio-Rad) and photographed using a ChemiDoc apparatus (Bio-Rad).