Mini problott membranes

To test the quality and specificity of the sera produced against the PHB recombinant proteins, we loaded 20 μg of protein from different purification samples and mixed with 2× Laemmli sample buffer [15 ]. Samples were loaded on 12.5% PGE-SDS gels and afterwards the electrophoresis gels were stained with Coomassie blue or silver nitrate [16 (link)]. Protein bands were transferred onto a PVDF membrane (Mini ProBlott Membranes, Applied Biosystems, Foster City, CA, USA) using a Bio-Rad transblot. Membrane strips were then incubated for 2 h at 37 °C with diluted 1:50 sera and, after several washes with PBST (PBS + Tween20), they were incubated for 2 h at RT with rabbit anti-mouse IgG (whole molecule)-peroxidase antibody (Sigma Aldrich, St. Louis, Missouri, USA) for Leish r PHB1 or with goat anti-rabbit IgG (whole molecule)-peroxidase antibody (Sigma Aldrich) at 1:1,000 for Leish r PHB2. Samples were developed using diaminobenzidine tetrahydrochloride (Sigma Aldrich) as a substrate (0.05% w/v) and H₂O₂ (dilution 1/5000).

To test the quality and specificity of the sera produced, we loaded 2 and 10 μg of the recombinant protein and total protein from cell culture samples, respectively, mixed with 2 × Laemmli sample buffer, on two 12.5% SDS-PAGE gels, one was stained them with Coomassie blue after electrophoresis, the other one was used to transferprotein bands onto a PVDF membrane (Mini ProBlott Membranes, Applied Biosystems, Foster City, CA, USA) using a Transblot device (Bio-Rad). Membrane strips was subsequently blocked for 30 min at room temperature in a TBS solution containing 1% casein and 0.05% Tween 20. After blocking, the membranes were incubated with chicken anti-rCSUI_001473 polyclonal sera, or negative chicken sera dilutions 1:500 in TTBS buffer (100 mM Tris, 0.9% NaCl, 0.1% Tween 20) at room temperature for 1 h. After rinsing with TTBS for 30 min, blots were exposed to biotinylated goat anti-chicken IgY (Vector Laboratories, Burlingame, CA, USA) as secondary antibody at 1:5000 dilution in TTBS buffer for 1 h at room temperature, incubated with avidin–biotin complex solution (Vector Laboratories) and finally detected by addition of 3,3′-5,5′-tetramethylbenzidine according to the manufacturer’s instructions (Vector Laboratories).