Goat anti mouse igg whole molecule alkaline phosphatase

Fifteen μg each of soluble and insoluble parasite crude worm extract and 100 ng each of recombinant OvCALR, MmCALR, and SjGST-OvCALR C-domain were size-separated by 12.5% Tris-glycine SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) by semi-dry blotting. Unspecific binding sites on the membranes were blocked in 5% skim milk (Thermo Fisher Scientific) in Tris-buffered saline (TBS, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5) at room temperature for 1 hr with gentle shaking. Mouse anti-rOvCALR antiserum and mouse pre-immune serum were diluted 1:3,000 in 1% skim milk in TBS. Each membrane was submerged in one of the antibody dilutions and incubated at 4°C with gentle shaking, overnight. On the next day, the membranes were washed 3 times for 5 min each in washing buffer (TBS, 0.05% Tween-20) and then incubated with goat anti-mouse IgG (whole molecule)-alkaline phosphatase (Sigma) at dilution 1: 30,000 in 1% skim milk in TBS at room temperature with gentle shaking, 1 hr. Following 3 washes in washing buffer for 5 min each, the membranes were equilibrated in detection buffer (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, and 50 mM MgCl₂) for 5 min. Finally, BCIP/NBT substrate (Amresco LLC, Solon, Ohio, USA) was added, and the membranes were incubated in the dark until the signals were obtained.

A new gel was prepared following the same steps described earlier. After electrophoresis, proteins were transferred to a 0.45 µm nitrocellulose membrane (BioRad Laboratories) at 100 V for 18 hours at 4°C. After that, the membrane was stained with Ponceau-S and discolored with PBS. The membrane was blocked with skimmed milk (La Sereníssima) 5% for 2 hours, at room temperature. mAb was diluted at 1:2000 in skimmed milk 2.5% and incubated overnight at 4°C. Then, the membrane was washed five times with PBS pH 7.2 and incubated with goat anti-mouse IgG (whole-molecule)-alkaline phosphatase (Sigma Aldrich) diluted at 1:5000 in skimmed milk 2.5%, for 2 hours, at room temperature. The membrane was washed again and the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT-plus) (Mabtech) was incubated for 20 minutes, at room temperature and protected from light. The reaction was stopped by adding distilled water and was considered positive by the appearance of color in the membrane.^{22 (link)}

Dot-ELISA was conducted pipetting 1 µL of whole cell suspension in nitrocellulose membrane 0.45 μm (BioRad Laboratories). After that, Ponceau-S staining was proceeded. Membranes were blocked with skimmed milk (La Sereníssima) 5% for 2 hours at room temperature. mAbs, diluted at 1:2000 in skimmed milk 2.5%, were incubated overnight at 4°C. Membranes were washed five times with PBS pH 7.2 and incubated for 2 hours, at room temperature, with goat anti-mouse IgG (whole-molecule)-alkaline phosphatase (Sigma Aldrich) diluted at 1:5000 in skimmed milk 2.5%. After washing, membranes were incubated with the substrate BCIP/NBT-plus (Mabtech) for 20 minutes, at room temperature and protected from light. We added distilled water to stop the reaction, which was considered positive by the appearance of color in the membrane.^{4 (link)}This project was performed at the Immunology Center of Adolfo Lutz Institute and was approved by the Technical and Scientific Committee of this institution (CTC number 41D-2011).