Hrp conjugated goat anti rabbit immunoglobulins

The presence of porcine CRT in cell lysates was confirmed by Western blotting using primary rabbit monoclonal antibody directed against CRT (Cell Signaling, D3E6, XP® Rabbit mAb #12238) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulins (DAKO, #00077731).
The binding of the FimH proteins to cell lysates was analyzed by far-Western blotting. Cell lysates or purified cell surface proteins separated by SDS-PAGE in 12% gel were transferred to nitrocellulose (Bio-Rad), blocked overnight with 3% BSA in TBST (TBS supplemented with 0.1% Triton X-100) and incubated with FimH proteins (0.1 mg/ml) for 4 h at RT. Immunodetection of FimH proteins was performed using primary rabbit polyclonal antibodies against FimH (Kisiela et al., 2005 (link)) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulins (DAKO, #00077731).

Cells (1 × 10⁶) were detached using a plastic cell scraper and solubilized in 100 µL of lysis buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, containing 1 mM ethylenediaminetetraacetate (EDTA), 0.5% NP40, and 1 mM phenylmethylsulfonyl fluoride (Roth, Karlsruhe, Germany)). The soluble proteins were quantified by the bicinchoninic method (Sigma, St. Louis, MO, USA). Cell lysates were subjected to vertical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (10% gel). Separated proteins were transferred to the PVDF membrane, and blotted proteins were incubated with the primary mAb against NUCB2 (Novus) at 4 °C overnight. After washing, the blots were incubated with HRP-conjugated goat anti-rabbit immunoglobulins (Dako) for 1 h at room temperature.