Horseradish peroxidase conjugated goat anti mouse igg

Protein samples were analyzed by SDS-PAGE with 10% or 12.5% gels [68 (link)]. Proteins were transferred from the gels to PVDF membranes using the Trans-Blot Turbo system (Bio-rad). Western blot analysis was performed with a monoclonal antibody raised against the Flag-epitope (Sigma) diluted 1:10,000 in a solution of PBS, 0.2% Triton, 3% skimmed milk and incubated for 16 hours at 4°C. Membranes were washed for 20 minutes each with PBS, 0.2% Triton solution. The washing step was repeated three times. Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Promega) diluted 1:2500 in a solution of PBS, 0.2% Triton for 1 hour at room temperature. Again, three washing steps of 45 minutes with PBS, 0.2% Triton solution were performed, and detection was performed by enhanced chemiluminescence using ImageQuant LAS54000 imaging system software (GE Healthcare Lifesciences).

Whole-cell protein extracts were prepared in Laemmli buffer (glycerol 5% vol/vol, β-mercaptoethanol 2.5%, SDS 1.15% p/v, Tris–HCl 31 mM pH 6.6 and bromophenol blue 0.05%). Protein samples were analyzed by Tris-Glycine-SDS triphasic gels with 16.5% polyacrylamide. Proteins were transferred from the gels to PVDF membranes by using a semidry electrophoretic transfer cell (Bio–Rad) at 15 V for 40 min. For Western blot analysis, a monoclonal antibody directed against the Flag epitope (Sigma–Aldrich) or a polyclonal antibody directed against the IrmA protein were diluted 1:10.000 or 1:1000 in a solution of PBS, 0.2% Triton and 3% skimmed milk. The membranes containing the proteins were incubated with the diluted antibody for 16 h at 4°C. The membranes were then washed for 10 min with PBS and 0.2% Triton X-100 (Sigma–Aldrich) solution. The washing step was repeated three times. Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Promega) or anti-rabbit IgG (Promega) diluted 1:2.500 in a solution of PBS and 0.2% Triton X-100 for 45 min at room temperature. Again, three washing steps with PBS and 0.2% Triton solution were performed for 30 min each, and immunodetection of the specific protein was performed by enhanced chemiluminescence by using the Molecular Imager ChemiDoc XRS system and Quantity One software (Bio–Rad).

Protein samples were analyzed by SDS-PAGE at 12.5% (Sambrook, 2001 ). Proteins were transferred from the gels to PVDF membranes using the Trans-Blot Turbo system (Bio-Rad). Western blot analysis was performed with monoclonal antibody raised against the Flag-epitope (1:10.000 – Sigma) incubating 16 h at 4°C. Membranes were washed three times of 20 min each with PBS 0.2% Triton solution. Thereafter they were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:2500 – Promega) during 1 h at room temperature. Again, membranes were washed three times of 20 min with PBS 0.2% Triton solution and detection was performed by enhanced chemiluminescence using Quantity One software (Bio-Rad).

Polystyrene, 96-well, flat-bottom plates (MaxiSorp, Nunc) were coated with SARS-CoV 2 B.1.617.2 Spike (R&D System) recombinant protein (0.05 µg in Carbonate–Bicarbonate Buffer; 100 per well) via overnight incubation at 4 °C. The wells were blocked with PBS containing 0.05% Tween-20 and 3% non-fat milk (PBST-milk) and incubated for 1 h at room temperature. Mouse sera and BALF were diluted in PBST-milk at 1:400 and 1:100, respectively. A total of 30 µL of undiluted nasal wash or 50 µL of diluted mouse sera or BALF was incubated for 2 h at 37 °C. Horseradish peroxidase-conjugated goat anti-mouse IgG (Promega Corporation, Madison, WI, USA) & IgA (Invitrogen, ThermoFischer Scientific, Waltham, MA, USA) was used to detect mouse antibodies bound to antigen-coated wells. Reactions were developed using 3,3′,5,5′-tetramethylbenzidine substrate (BioFX™) and terminated using 1M HCl. Absorbance was measured at 450 nm.

Protein extracts were prepared in Laemmli buffer⁷⁵ (5% glycerol, 2.5% β-mercaptoethanol, 1.15% SDS, Tris-HCl 31 mM pH 6.6 and 0.05% bromophenol blue). Protein samples were analyzed in 16.5% polyacrylamide Tris-tricine-SDS triphasic gels. After electrophoresis, the proteins were transferred from the gels to PVDF membranes using a semidry electrophoretic transfer cell (Bio-Rad) at 15 V for 40 min. For the Western blot analysis, a monoclonal antibody directed against the Flag epitope (Sigma-Aldrich) diluted 1:10,000 in a solution of PBS, 0.2% Triton and 3% skim milk was used. The membranes containing the proteins were incubated overnight with the diluted antibody at 4 °C. After incubation, the membranes were washed three times for 10 min with a solution of PBS and 0.2% Triton X-100 (Sigma-Aldrich). Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Promega) diluted 1:2500 in a solution of PBS and 0.2% Triton X-100 for 45 min at room temperature. After incubation, three additional washing steps with PBS and 0.2% Triton solution were performed for 30 min each time. The immunodetection of the specific protein was performed by enhanced chemiluminescence using a Molecular Imager ChemiDoc XRS system and Quantity One software (Bio-Rad).