2.3 Western Blotting
B. canis RM6/66 (ATCC 23365) proteins were dissolved in SDS-PAGE denaturing buffer (Life Technologies), loaded into NuPAGEĀ® 4-12% Bis-Tris gels (Life Technologies) and separated at constant voltage (200 V). Then, proteins were blotted on nitrocellulose membranes (Life Technologies) using iBlot2 Dry Blotting System (Life Technologies) at 20 V for 1 min, 23 V for 4 min and 25 V for 2 min. Membranes were blocked with 5% skim milk in 0.01 M PBS, pH 7.2, containing 0.05% Tween 20 (PBST) for 2 h at RT. Then, membranes were incubated overnight (ON) at RT with canine sera diluted 1:5000 in PBST containing 2.5% skim milk. After three washes with PBST for 10 min, membranes were incubated for 1 h at RT with Protein A-HRP (Sigma) diluted 1:5000 in PBST containing 2.5% skim milk. After three washes with PBST and one final wash with PBS for 10 min, immune complexes were detected by chemiluminescence (ECL Western Blotting Detection Kit, GE Healthcare) using the Chemidoc MP (Bio-Rad); analyses were performed using Image Lab Software version 4.0.1 (Bio-Rad).