Hrp conjugated anti his monoclonal antibody

Purified recombinant S. pseudintermedius Sbi proteins (prSbi) were separated on 7.5% polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (Thermoscientific). Blots were treated overnight with blocking buffer (BB) containing 5% powdered skim milk and 0.05% polyoxyethylene sorbitan monolaurate (Tween 20) in PBS, pH 7.2 at 4°C. Blocked membranes were incubated with horseradish peroxidase (HRP) -conjugated anti-HIS monoclonal antibody (Thermoscientific, 1:2000 dilution) for 1 h at room temperature followed by three washes with TBST buffer (PBS containing 0.05% Tween20). Reactive bands were visualized using 4-chloro-1-naphthol solution (1-Step,Thermo Scientific).
Binding of prSbi to dog IgG was determined using western blots (prepared and blocked as described above) incubated with dog IgG whole molecule (Rockland Antibodies and Assays) diluted 1:1000 and detected with HRP conjugated goat anti-dog IgG (H+L chain, Bethyl), diluted 1:3000.

Samples were subjected to SDS–PAGE gel, and the proteins were transferred to PVDF membrane. Membrane was blocked with 2% bovine serum albumin (BSA) in TBST (50 mM Tris–Cl, 150 mM NaCl, pH 8.0%, and 0.05% Tween 20) for 1 hr. The blot was then incubated with HRP-conjugated anti-His monoclonal antibody (1:5000) and detected with supersignalfemto substrate (Thermoscientific, USA) using ChemiDoc imaging system (Bio-Rad Laboratories, USA).