Anti rabbit igg hrp linked secondary antibody

The production of PilA, which is a type IV pilin produced by A. nosocomialis (Harding et al., 2013 (link)) was determined by western blotting using anti-PilA polyclonal rabbit antisera generated as described (Harding et al., 2015 (link)). Briefly, cells of the ATCC 17978, NFAb-1 and NFAb-2 strains were collected from the plastic surface of motility plates after the agarose layer of MMA plates was removed, while ATCC 19606^T cells, which do not display twitching motility, were collected from the surface of MMA plates. Cells were resuspended in sterile PBS and OD₆₀₀ values were determined to normalize cell numbers prior to lysis. Cell pellets were then lysed in 100 µl of 5X SDS sample buffer (250 mM Tris-HCl, pH6.8; 10% SDS; 30% glycerol; 5% beta-mercaptoethanol and 0.02% bromophenol blue) and boiled for 5 minutes. Equal volumes of cell lysates were loaded onto 4-12% NuPAGE Bis-Tris gels (Invitrogen), transferred to Immobilon-PSQ PVDF membranes (Millipore). Membranes were incubated at 4°C with a 1:1,000 dilution of anti-PilA serum for 16 h. Immunocomplexes were detected by chemiluminescence using an 1:5,000 dilution of anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare) and ECL Prime Western Blotting Detection Reagent (GE Healthcare).

Cell lysates were prepared 48 h post-transfection in 1 × Na Page sample buffer (FUJIFILM Wako Pure Chemical Industries). Cell lysate proteins (5 μg/well) were separated by iBlot (Thermo Fisher SCIENTIFIC). CDT1 expression was analyzed by Western blotting. The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Dulbecco’s Modified Eagles Medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin (P/S), Opti-MEM I (OMEM), anti-goat IgG HRP-linked secondary antibody, Simple Blue Stain, Novex 4–12% Tris-Glycine SDS-PAGE gels, dithiothreitol (DTT) Immulon-2HB 96-well plates, Nickel coated 96-well plates and Lipofectamine 2000 transfection reagent were all purchased from Thermo Fisher Scientific. Zanamivir and 2’-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) were acquired from Moravek Inc and Cayman Chemicals, respectively. β-propiolactone, formaldehyde, o-Phenylenediamine dihydrochloride (OPD), and anti-mouse IgG HRP linked secondary were purchased from Sigma. Anti-rabbit IgG HRP-linked secondary antibody and 0.45-μm polyvinylidene difluoride (PVDF) membrane were obtained from GE healthcare. Specific-Pathogen-Free (SPF) eggs and turkey red blood cells (TRBCs) were purchased from Charles River Labs and the Poultry Diagnostic and Research Center (Athens, GA), respectively. The H1N1 A/Brisbane/02/2018 field isolate (WT) and CVV (IVR-190) were kindly provided by the WHO. Rabbit Antisera against NA was generated by Agrisera (Sweden) using NA-WSN residues 35–453 isolated from E. coli inclusion bodies [29 (link)]. Polyclonal goat antiserum against the H1N1 influenza virus A/Fort Monmouth/1/1947 (NR-3117) was obtained from BEI Resources, NIAID, NIH.