Goat anti mouse igg1 hrp

Macrophages were lysed in RIPA buffer supplemented with 4 mM Na₃VO₄, 10 mM NaF and 1:100 protease inhibitor (Sigma Aldrich). Protein concentrations were determined by BCA assay. Cell lysates were denatured with 2-mercaptoethanol and incubated at 95 °C for 8 min, 20ug protein was loaded on a 12% SDS-PAGE gel and blotted on a blot membrane (Millipore). The blot was cut in two before incubation with the antibodies. Blots for phospho- IκBα were incubated in blocking buffer (Tris buffered saline supplemented with 0.1% Tween 20 (Sigma Aldrich)) containing 5% milk or for total IκBα in blocking buffer with 5% BSA for 1 hour). Blots were incubated with primary mouse anti-phospho-IκBα (Cell Signaling) 1:1000 antibody in blocking buffer o/n. As secondary antibody, goat anti-mouse IgG1-hrp (1:1000 in blocking buffer) (Sigma Aldrich) was used. For total IκBα, rabbit anti-mouse polyclonal IκBα (1:500) (Santa Cruz) and as secondary antibody goat anti-rabbit-hrp (1:1000) (Sigma Aldrich) was used. Samples were detected with ECL+ (Amersham) using autoradiography films (GE Healthcare). Western blot band were quantified with Image J software.

The VLP-specific IgG, IgM, IgG1 and IgG2a responses in the serum from antigen-immunized mice were analyzed by sandwich ELISA. Ninety-six-well plates were coated with 1 μg/mL VLP at 4 °C for 24 h. The plates were washed 5 times with PBS containing 0.2% Tween 20 (PBS-T), and then blocked with 5% BSA in PBS at RT for 1 h. After blocking, diluted serum was added to each well and incubated at RT for 1 h. Unbound antibodies were removed by washing with PBS-T, and then goat anti-mouse IgG-HRP (Sigma-Aldrich), goat anti-mouse IgM-HRP (Sigma-Aldrich), goat anti-mouse IgG1-HRP (Sigma-Aldrich), or IgG2a-HRP (Sigma-Aldrich) were added to the wells, and incubated for 30 min at RT. After washing the plates 5 times with PBS-T, 100 μL of TMB substrate reagent (BD Biosciences, Franklin Lakes, NJ, USA) was added. When colors developed, 50 μL of 2 N H₂SO₄ was added, and optical absorbance was measured at 450 nm using a Victor X3 light plate reader (Perkin-Elmer, Waltham, MA, USA).