Anti 6x his tag monoclonal antibody

The recombinant baculovirus-infected Sf9 cells were collected 48 h post infection (hpi). The cell lysates were re-suspended in equal volumes of a 2× Laemmli sample buffer (Bio-Rad, USA), separated by 12% SDS-PAGE, and transferred onto nitrocellulose membranes using a semi-dry transfer cell (Bio-Rad, USA). The membranes were then blocked with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.5% Tween 20) containing 5% skim-milk powder. The recombinant VP1 (rVP1) was detected by Western blotting, using an anti-6X His tag^® monoclonal antibody (Abcam, USA) or chicken anti-CAV polyclonal antibodies as the primary antibodies. The expression of rVP2 was also verified by polyclonal antibodies recognizing E. coli-expressed VP2 as the primary antibody. In addition, the detection of recombinant chIL-12 by Western blotting was performed with anti-6X His tag^® monoclonal antibody (Abcam, USA) or polyclonal antibodies recognizing E. coli-expressed chIL-12 as the primary antibodies [27 (link)]. Alkaline phosphatase-conjugated goat-anti-mouse or -chicken IgG (KPL, USA) were used as the secondary antibodies to label the protein bands.

Sf9 cells were cultured in six-well plates (Nunc, USA) at a density of 1.5 × 10⁶/mL, and infected with one of the recombinant baculoviruses, B-VP1/B-VP2 or B-chIL-12, at a multiplicity of infection (MOI) of 1. At 3 dpi, the infected cells were fixed with a 1:1 mixture of acetone and methanol for 30 min at 4 °C. After washing with phosphate-buffered saline (PBS) three times, the fixed cells were treated with chicken anti-CAV polyclonal antibodies, anti-6X His tag^® monoclonal antibody (Abcam, USA), or antibodies recognizing E. coli-expressed chIL-12 as the primary antibodies. Alex Fluor 594-conjugated goat anti-chicken IgG H&L (Abcam, USA) or Alex Fluor 594-conjugated goat anti-mouse IgG H&L (Abcam, USA) was used as the secondary antibody for the B-VP1/B-VP2 infected Sf9 cells to detect rVP1, respectively. The cells infected by B-chIL-12 were detected with the Alex Fluor 555-conjugated goat anti-mouse IgG H&L (Abcam, USA) as the secondary antibody. The cells were visualized using a fluorescence microscope (DM IRB, Leica, Germany).