Peroxidase conjugated goat anti mouse igg antibody

To determine anti-PE or anti-PilA IgG levels, microtiter plates were coated with PE or PilA (2 μg/ml or 4 μg/ml, respectively, in carbonate buffer) overnight at 4°C. After washing, serial 2-fold dilutions of murine sera (starting at 1/500 for the PE assay or 1/20 to 1/500 for the PilA assay, depending on the experiment, in phosphate-buffered saline [PBS] containing 0.05% Tween 20 [PBS-T] for 1 h at 25°C).
Afterwards, in both assays, peroxidase-conjugated goat anti-mouse IgG antibodies (1/2,500 or 1/1,250 in PBS-Tween; code 115-035-003; Jackson Laboratories) were added for 1 h at 25°C. The colorimetric reaction was obtained by the addition of o-phenylenediamine dihydrochloride in citrate buffer in the presence of hydrogen peroxide for 15 min and stopped by the addition of 1 N HCl. The plates were read in a spectrophotometer at 490 and 620 nm. In both cases, an in-house-calibrated reference serum sample was used, and IgG concentrations (expressed in micrograms per milliliter) were calculated by the 4-parameter method using Soft Max Pro software.

The antibodies used for immunoblotting and immunostaining are as follows: anti-myc (MC045, Nacalai Tesque, Japan), anti-Flag M2 (Sigma-Aldrich), fluorescein isothiocyanate-conjugated goat anti–mouse F(ab’)² (Cappel Laboratories, Durham, NC, United States), peroxidase-conjugated goat anti–mouse IgG antibodies (Jackson Immuno Research Laboratories, West Grove, PA, United States). For western blotting, the cells were lysed in CelLytic M Cell Lysis Reagent (Sigma-Aldrich) containing a protease inhibitor cocktail (Roche). The lysates were boiled with SDS-sample buffer at 95°C for 3 min. The samples were subjected to SDS-PAGE, transferred to PVDF membranes by iBlot system (Invitrogen), and incubated with primary antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. Finally, chemiluminescence was detected using Chemi-Lumi One Super kit (Nacalai Tesque), and luminescence images were analyzed by LAS 4000 (GE Lifesciences). Immunostaining of HeLa cells with anti-myc antibody was performed as described previously (Kataoka et al., 2011 (link)).