Goat anti mouse ig alkaline phosphatase conjugate

One microgram each of the full-length recombinant S1 (rS1), S1-0, S1-A, and S1-BCD were prepared in protein loading buffer and subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were blotted onto the nitrocellulose membrane (NC). The empty sites of blotted NC were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h. After washing with TBST, the NC was immersed into a solution of either mouse anti-6× His antibody or purified mAbG3 solution (5 μg in 6 ml TBST) and kept at room temperature for 1 h. The membrane was washed with the TBST and placed in a solution of goat anti-mouse Ig-alkaline phosphatase (AP) conjugate (1:3,000; Southern Biotech, Birmingham, AL, United States) in TBST at room temperature for 1 h. After washing, BCIP/NBT substrate (KPL, Gaithersburg, MD, United States) was used for the visualization of the antigen–antibody reactive bands. The enzymatic reaction was stopped by rinsing the NC with distilled water.

The antigenic preparation was electrophoresed in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) using 4% stacking gel and 12% separating gel cast in Mini-PROTEAN Cell (Bio-Rad, Hercules, California, USA). The SDS-PAGE-separated components in the separating gels were electroblotted onto nitrocellulose membrane (NC) and the unoccupied sites on the blotted NC were blocked with 5% skim milk in Tris-buffered saline containing 0.01% Tween-20 (TBS-T) for 1 h. Excess blocking reagent was discarded; the NC was washed three times with the TBS-T and placed in a solution of mouse mAb to HuscFv34, mouse anti-His (Bio-Rad), or mouse anti-SUMO (Genscript) at RT for 1 h. The NC was washed again with the TBS-T and incubated with goat-anti-mouse Ig-alkaline phosphatase (AP) conjugate (SouthernBiotech, Birmingham, AL, USA) for 1 h. After washing with TBS-T, BCIP/NBT substrate (KPL, SeraCare, Millford, MA, USA) was used to reveal the antigen-antibody reactive bands. The NC was washed with distilled water and air-dried.