Peroxidase conjugated anti goat igg

Convertase C3bBb stabilization experiments were done as described before (9 (link)). In short, 2.5 × 10⁷ cells of S. aureus Wood were incubated for 20 min at 37 °C in veronal-buffered saline, pH 7.4, containing 1 mm MgCl₂ and 1 mm CaCl₂ plus 0.1% (v/v) BSA with different concentrations of horse serum and 1 μm SCIN-A, eqSCIN, or buffer control). After centrifugation, bacterially associated proteins were separated by SDS-PAGE, followed by immunoblot. Horse Bb was detected with goat anti-human factor B (Complement Technology) followed by peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology, Inc.).

Protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Same concentrations of protein from colon or liver extracts were loaded to perform electrophoresis. More precisely, equivalent amounts of proteins were boiled in loading buffer containing 4% SDS, 20% glycerol, and bromophenol blue for 5 minutes. Proteins were resolved on 10% SDS-PAGE gels and then were transferred onto nitrocellulose membranes (GE Healthcare; Amersham Biosciences, Baie d’Urfé, QC, Canada). The membranes were blocked with 5% non-fat dry milk solution and incubated with antibodies against lipocalin 2 (R&D Systems, Minneapolis, MN, USA), MyD88 (Cell Signaling, Danvers, MA, USA) and β-actin (Abcam, Cambridge, MA, USA). To detect the formation of immunocomplexes, peroxidase-conjugated anti-goat IgG (Santa Cruz, Dallas, TX, USA) and anti-mouse IgG (R&D Systems, Minneapolis, MN, USA) were used as secondary antibodies. Staining intensity was developed with an Amersham enhanced chemiluminescence system (GE Healthcare, Amersham Biosciences, Baie d’Urfé, QC, Canada).

Cells were lysed on ice in lysis buffer. The lysates were then boiled for 5 min, clarified by centrifugation at 15,000 × g for 15 min, and separated by SDS-PAGE. The proteins were then transferred onto nitrocellulose membranes, which were incubated with a 1:100–200 dilution of human polyclonal or monoclonal antibodies raised against the following: E-cadherin, N-cadherin, pERK, ERK (Santa Cruz, CA, USA), fibronectin(R&D, MN, USA), CD133 (MiltenyiBiotec, Germany), and pSRC (CST, MA, USA). Next, a 1:200–1000 dilution of peroxidase-conjugated anti-goat IgG, anti-rabbit IgG (Santa Cruz, CA, USA), or anti-mouse IgG (Jackson ImmunoResearch, PA, USA) antibody was applied for the secondary reaction. As an internal control for protein loading, β-actin was detected using a specific antibody (Sigma, MO, USA). Immune complexes were visualized using the ECL Western blotting detection system (Amersham, UK).