Binding assays were performed as described (28 (link), 40 (link)). Briefly, 96-well plates (Nunc) were preadsorbed with Vn-HX (5 μg/ml), Vn (5 μg/ml; Sigma), or gelatin (20 μg/ml; Sigma), then blocked with tris-buffered saline (TBS) containing 3% milk, and washed with TBS supplemented with 0.05% Tween 20. Recombinant C-terminal His-tagged Ail (Ail-His) was purified, refolded, and concentrated to 80 μg/ml in buffer with 4 mM decylphosphocholine and then added in decreasing concentrations to the preadsorbed wells. After incubating overnight at 4°C, bound Ail-His was detected with mouse anti-His monoclonal antibody (Qiagen), secondary goat anti-mouse antibody conjugated to horseradish peroxidase (Sigma), and the horseradish peroxidase substrate o-phenylenediamine (Pierce).
Goat anti mouse antibody conjugated to horseradish peroxidase
Manufactured by Merck Group
The Goat anti-mouse antibody conjugated to horseradish peroxidase is a laboratory reagent used as a detection antibody in various immunoassay techniques. It consists of a goat-derived antibody specific to mouse immunoglobulins, covalently linked to the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti his monoclonal antibody, by Qiagen (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by Cytiva (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
2 protocols using goat anti mouse antibody conjugated to horseradish peroxidase
1
Ail Protein Binding Assay
2
Immunoblot Analysis of VSVΔG-HA-ZGP Expression
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Expression of HA and ZGP from VSVΔG-HA-ZGP was confirmed by immunoblotting. Vero E6 cells infected with VSVΔG-HA-ZGP was harvested at 48 hours postinfection along with cell supernatant, and centrifuged at 2500 rpm. After removal of excess supernatant, the virus lysate was combined with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) buffer containing 250 mM Tris–HCl pH 6.8, 30% glycerol (volume/volume), 8% SDS, 0.02% bromophenol blue, and 10% 2-β-mercaptoethanol (volume/volume). Proteins were separated by electrophoresis on a 10% polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare) overnight. The HA and ZGP antigens were detected using mouse serum containing anti-H5N1 HA or a monoclonal mouse anti-ZGP antibody, respectively, at 1:1000. The control antigen was detected using a monoclonal mouse anti-β-actin at 1:5000 (Sigma). Goat antimouse antibody conjugated to horseradish peroxidase (Sigma) was used at 1:30000 as a secondary antibody. Bands were visualized using the enhanced chemiluminescence detection kit following manufacturer's instructions (Amersham).
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