Horseradish peroxidase hrp conjugated anti mouse igg antibody

To detect the pilus major subunit FctA, S. pyogenes and L. lactis cells were grown at 37°C or 25°C until the late exponential phase (OD₆₀₀ = 1.0 for S. pyogenes; 0.5 for Lactococcus), then washed with PBS and suspended in protoplast buffer containing 0.1 M KPO₄ (pH 6.2), 40% sucrose, 10 mM MgCl₂, complete EDTA‐free protease inhibitors (Roche) and 250 µg/ml N‐acetylmuramidase SG (Seikagaku Biobusiness). The suspension was incubated at 37°C for 3 h and protoplasts were sedimented by centrifugation at 20,000 × g for 20 min. Proteins in the supernatant were separated in a 5–12% acrylamide gel by sodium dodecyl sulfate‐poly‐acrylamide gel electrophoresis (SDS‐PAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked overnight using a casein‐based blocking reagent (Megmilk Snow Brand) at 4°C and incubated at room temperature (RT) for 1 h with mouse antisera diluted 1:2,000 in Tris‐buffered saline (TBS) containing 0.2% Tween 20 (TBST). After washing three times with TBST, each membrane was incubated at RT with horseradish peroxidase (HRP)‐conjugated anti‐mouse IgG antibody (Cell signaling), diluted at 1:2,000 in TBST for 1 h. The membranes were then washed three times and developed using the Pierce western blotting substrate (Thermo Scientific) or 3,3′,5,5′‐tetramethyl‐benzidene colorimetric substrate (MOSS).

Antigens were diluted in Tris-buffered saline (TBS, pH 7.4) and coated onto Maxisorp 96-well plates (Thermo Fisher Scientific) at 100 ng/well. After blocking with 5% skim milk in TBST (TBS with Tween-20), primary antibodies were added and incubated for 2 h at 37°C. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Cell Signaling Technology, Danvers, MA, USA) or anti-human polyvalent immunoglobulins (G,A,M)-peroxidase antibody (Sigma-Aldrich) was used as the secondary antibodies. The antigen-antibody reaction was visualized with Ultra-TMB solution (Thermo Fisher Scientific). After 10 min, 0.2 M H₂SO₄ was added to stop color development. The color reaction was measured at 450 nm (OD₄₅₀) with a VersaMax microplate reader (Molecular Devices, San Jose, CA, USA).