Horseradish peroxidase anti rabbit immunoglobulin g conjugate

Fractionation of L. interrogans strain RJ19115 to enrich for outer membrane (OM) proteins was performed using Triton X-114 as previously described (Nally et al., 2001 (link)). OM enriched fractions were compared to whole leptospires by 1-D gel electrophoresis as previously described (Monahan et al., 2008 (link)). Proteins were visualized by staining with Sypro Ruby (Invitrogen, CA) and lipopolysaccharide was visualized by staining with Pro-Q Emerald 300 (Invitrogen, CA) as per manufacturer's guidelines. For immunoblotting, samples were transferred to Immobilon-P transfer membrane (Millipore, 220 Bedford, MA) and blocked overnight at 4°C with StartingBlock (TBS) blocking buffer (Thermo Scientific, CO). Membranes were individually incubated with indicated antisera (anti-LipL21, anti-LipL32 and anti-LipL41 at 1:4,000, anti-Treponema FlaA at 1:2,000, or a pool of sera from infected or non-infected rats at 1:1,000, in PBS-T for 1 h at room temperature), followed by incubation with horseradish-peroxidase anti-rabbit immunoglobulin G conjugate or horseradish-peroxidase anti-rat immunoglobulin G conjugate (Sigma, MO). Bound conjugates were detected using Clarity Western ECL substrate (BioRad, CA) and images acquired using a Bio-Rad ChemiDoc MP imaging system.

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.