Vertical electrophoretic minicell unit

SDS-PAGE was performed on a 12% polyacrylamide gel using a discontinuous buffer system. The extracted LPS were mixed with 2X SDS-PAGE sample loading buffer (BioRad, Hercules, CA, USA) and boiled for 5 min before loading. Electrophoresis was carried out in a vertical electrophoretic minicell unit (BIORAD, Hercules, CA, USA) for 2 h at 120V in Tris-glycine running buffer (25mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3). The separated LPS were transferred to nitrocellulose membranes (0.2 μm pores size; Schleicher and Schuell, Keene, NH) [17 (link)] and blocked with 4% non-fat dry milk in Tris-buffered saline (20mM Tris, 150mM NaCl, 0.05% Tween 20, pH 7.5;TBST). Membranes were incubated with serum (1:200) from patients positive for leptospirosis, followed by incubation with anti-human IgG antibody conjugated with horseradish peroxidase (Sigma, St. Louis, Mo) and bands visualized by using 4-chloro-α- naphthol (Sigma, St. Louis, Mo).

The purity of the proteins was checked using SDS-PAGE electrophoresis in a vertical electrophoretic mini-cell unit (Bio-Rad, Hercules, CA), in Tris-glycine running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS [pH 8.3]), for 1 h at 120 V. Proteins were transferred to an Immobilon-P PVDF membrane (pore size, 0.45 μm; Millipore Sigma, United States) and blocked with 5% non-fat dry milk in Tris-buffered saline (20 mM Tris, 150 mM NaCl, 0.2% Tween 20 [pH 7.5]). Membranes were incubated with anti-SgGAPDH immune sera raised in rabbits, followed by incubation with secondary antibody (anti-rabbit IgG; Cell Signaling Technology, United States). Reacted protein bands were visualized by using Pierce^TM ECL 2 Western Blotting Substrate (Thermo Scientific, United States) and imaging.