Leptospires (mid-late log phase, 1–3 × 108 leptospires/mL) were harvested by centrifugation (10,000 × g, 4°C, 30 min), washed twice with PBS, and processed for one-dimensional (1-D) SDS-PAGE on 12% acrylamide gels (BioRad) as per manufacturer's guidelines. Proteins were visualized by staining with Sypro Ruby (Invitrogen, CA, USA) and lipopolysaccharide was visualized by staining with Pro-Q Emerald 300 (Invitrogen, CA) as per manufacturer's guidelines. For immunoblotting, samples were transferred by semi-dry transfer (Amersham TE77 PWR) to Immobilon-P transfer membrane (Millipore, 220 Bedford, MA) and blocked overnight at 4°C with Starting Block (PBS) blocking buffer (Thermo Scientific, CO) (25 (link)).
Membranes were individually incubated with indicated antisera diluted in blocking buffer (anti-LipL32 at 1:4,000, or anti-Alexi, anti-Hardjo at 1:1,000) followed by incubation with horseradish-peroxidase anti-rabbit immunoglobulin G conjugate diluted 1:4,000 in blocking buffer (Sigma, MO). Bound conjugates were detected using Clarity Western ECL substrate (BioRad, CA) and images acquired using a Bio-Rad ChemiDoc MP imaging system.
Membranes were individually incubated with indicated antisera diluted in blocking buffer (anti-LipL32 at 1:4,000, or anti-Alexi, anti-Hardjo at 1:1,000) followed by incubation with horseradish-peroxidase anti-rabbit immunoglobulin G conjugate diluted 1:4,000 in blocking buffer (Sigma, MO). Bound conjugates were detected using Clarity Western ECL substrate (BioRad, CA) and images acquired using a Bio-Rad ChemiDoc MP imaging system.
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