Rabbit anti influenza a virus nucleoprotein antibody

For IVIG fractions and their fragments, loaded 1 μg protein aliquots and Precision Plus protein standards (BIO-RAD, California, USA) onto 10% reducing SDS-PAGE gels. After electrophoresis, the separated proteins were transferred to nitrocellulose filter membranes. Rabbit anti-human IgG γ Fc region antibody (Dako, Copenhagen, Denmark), mouse anti-human Ig κ chainantibody (ZSGB-BIO, Beijing, China) and mouse anti-human Ig λ chain antibody (ZSGB-BIO, Beijing, China) at a dilution of 1:1,000 were used as primary antibodies.
For cell lysates of infected A549 or MDCK cells, they were prepared using RIPA buffer (Millipore, Massachusetts, USA), and the concentrations were determined with BCA protein assay kit (Thermo Scientific, Illinois, USA). About 20μg protein aliquots were separated and transferred to nitrocellulose filter membranes. Rabbit anti-Influenza A Virus Nucleoprotein antibody (GeneTex, California, USA) and mouse anti-β actin monoclonal antibody (ZSGB-BIO, Beijing, China) at a dilution of 1:1,000 were used as primary antibodies.
After incubation with appropriate secondary antibodies including Goat anti-rabbit IgG-680 (1:10,000; LI-COR, Nebraska, USA) and goat anti-mouse IgG-680 (1:10,000; LI-COR, Nebraska, USA), the blots were examined using a Odyssey imaging system (LI-COR, Nebraska, USA).

Western blot was performed essentially the same as previously described54 (link). Briefly, cells were lysed in RIPA buffer (Cell Signaling Technology, Boston, MA, USA) containing protease and phosphatase inhibitors (Roche, Penzberg, Germany). The concentrations of protein in the lysates were determined with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s protocol. After electrophoresis on 10% SDS-PAGE Bio-Rad minigels, the aliquots proteins were transferred to nitrocellulose filter membrane (Whatman/GE Healthcare life sciences, Marlborough, MA, USA). The blots were blocked for 1 hour at room temperature in 5% skim milk in TBST, and incubated overnight with rabbit anti-Influenza A Virus Nucleoprotein antibody (GeneTex, Irvine, CA, USA) and mouse anti-β actin monoclonal antibody (ZSGB-BIO, Beijing, China) at a dilution of 1:10,00 as primary antibodies. IRDye 680-conjugated goat polyclonal anti-rabbit IgG (H+L) and IRDye 800-conjugated goat polyclonal anti-mouse IgG (H+L) (both from LiCor, Lincoln, NE, USA) were used as secondary antibodies at a dilution of 1:10,000. The blot was examined and photographed with a Odyssey imaging systems (LiCor, Lincoln, NE, USA).