Ap conjugated polyclonal goat anti mouse igg

Female BALB/C mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. In the experiments outlined below, avidin from egg white (Aladdin), C1q (EMD chemicals), alkaline phosphatase (AP) substrate P-nitrophenyl phosphate (Sigma-Aldrich), Mouse total IgG (Sigma-Aldrich), and human total IgG (Sigma-Aldrich) were used. Furthermore, the following antibodies were adopted: AP-conjugated polyclonal goat anti-human IgG (γ-chain specific) (Sigma Aldrich), AP-conjugated polyclonal goat anti-mouse IgG (whole molecule, Sigma-Aldrich), AP-conjugated polyclonal goat anti-rabbit IgG (whole molecule, Sigma-Aldrich), and polyclonal rabbit anti-human C3c (Dako). C1q CLR was produced by partial pepsin digestion of C1q as previously described (13 (link)).

To analyze the frameshift expression of P3N-PIPO, total proteins were extracted from N. benthamiana leaves infected with PVX chimeric viruses using a lysis buffer (100 mM Tris-HCl (pH = 6.8), 20% SDS, and 2% β-mercaptoethanol) and were separated by 12% SDS-PAGE. Proteins were electroblotted onto a preactivated PVDF membrane. Then, the membrane was blocked with block buffer (50 mL 1 × TBS buffer containing 5% skimmed milk powder) and probed with the GFP monoclonal antibody (Sigma-Aldrich Corp., St. Louis, MO, USA) at room temperature for 2 h. Following three washes of 5 min each in 1 × TBS containing 0.1% Tween 20, proteins were incubated with alkaline phosphatase (AP)-conjugated polyclonal goat anti-mouse IgG (Sigma-Aldrich Corp., St. Louis, MO, USA) and visualized using the NBT/BCIP^® solution (Sigma-Aldrich Corp., St. Louis, MO, USA).