Hrp conjugated goat anti mouse igg fc

Standard procedures of Western blotting were used to determine the reactivity of the mAb or pAb to the whole cell lysates of S. eriocheiris, S. melliferum, S. mirum and U. urealyticum on a 12% gel7 (link). The mAbs or pAb we prepared were used as the primary antibody and the HRP conjugated goat anti-mouse IgG Fc or anti-rabbit as the secondary antibody (Chemicon, Japan). The culture medium of spiroplasmas or pre-immunized rabbit serum was used as the negative control.

3T3 cells were transiently transfected with AAV-CB-hACE2 plasmid by calcium phosphate transfection and lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% Sodium dodecyl sulfate, 50 mM Sodium fluoride) containing complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) at 72 hours post transfection. The cell lysate was then separated by 4–12% Bis-Tris gradient gel (NuPAGE, Thermo Scientific, IL, USA) and transferred to the PVDF membranes (Bio-Rad, CA, USA). The transferred membranes were blocked with 5% BSA in PBST (PBS containing 0.05% Tween-20) for an hour at room temperature and then incubated with either mouse anti-hACE2 (R&D, MN, USA) or mouse anti-actin antibody (Sigma-Aldrich, MO, USA) in the blocking buffer overnight at 4°C. HRP-conjugated goat-anti-mouse IgG Fc (Chemicon, CA, USA) was served as the secondary antibody. The blots were visualized with ECL western blot substrate (Millipore, MA, USA) and analyze by LAS 3000 (Fujifilm, Tokyo, Japan).