Hrp conjugated anti mouse igg

For the measurement of serum anti-human PG antibodies, wells of 96-well ELISA plates (Nunc) were coated overnight with human PG (1 μg/well each) in 100 μl/well of sodium carbonate buffer at room temperature [31 (link)]. Unbound antigen was removed by washing with 0.05% Tween 20 in PBS. Wells were blocked with 1.5% non-fat milk in 250 μl/well PBS for 1 h at room temperature on a shaker platform. Serum samples were diluted to 1:100 in blocking buffer and incubated with the antigen-coated wells (100 μl/well, duplicate wells) for 2 h at room temperature with shaking. Bound IgG was detected by incubation with 100 μl/well of HRP-conjugated anti-mouse IgG (BD Biosciences) at a 1:2000 dilution for 2 h at room temperature with shaking. Unbound material was removed with wash buffer between each of these steps. The color reaction was developed with 100 μl/well of O-phenylene-diamine solution (Sigma-Aldrich) dissolved freshly in PBS with H₂O_2, and incubation for 30 min in the dark. The reaction was stopped with 25 μl/well stop solution (4 N H₂SO₄). Absorbance at 450 nm was read in an ELISA reader.

The 3T3-L1 cells were lysed in RIPA buffer (Wako Pure Chemical Industries, Japan) supplemented with protease inhibitor cocktail (Roche) for 30 min on ice and centrifiguted at 10⁴ x g for 10 min to obtain cell lysates. The resultant lysate was run on 4–12% Tris-Glycine NuPAGE gels (Life Technologies) and transferred onto PVDF membrane (Bio-Rad). Membranes were blocked with 5% skimmed milk in PBS-T at room temperature for 2 h and incubated with rabbit anti-adiponectin (#2789S, Cell Signaling Technologies) or mouse anti-β-actin monoclonal (Clone AC-15, Sigma) at 4°C overnight. Membranes were washed 3X with PBS-T and incubated with HRP-conjugated anti-rabbit IgG (#7074, Cell Signaling Technologies) or HRP-conjugated anti-mouse IgG (BD Biosciences) in 5% skimmed milk in PBS-T at room temperature for 1hr. After washes 5 X with PBS-T, the bands were visualized by enhanced chemiluminescence (ECL, Perkin-Elmer) and quantified using by a CCD camera system (LAS4000mini, GE Healthcare) with ImageQuant TL software (GE Healthcare).