Bovine Mastitis Immune Response Profiling

Western blot was conducted as described by Abdi et al. [33 (link)]. Briefly, the SUSP were separated by electrophoresis and transferred to nitrocellulose membrane (Bio-Rad) at a constant 100 V with 400 mA for 60 min by a wet method (Bio-Rad). The membrane was blocked overnight, washed, and incubated with hyperimmune serum from a cow previously vaccinated with SUSP and protected from mastitis upon challenge in another previous pilot study. The membrane was washed and incubated with horse radish peroxidase (HRP)-conjugated sheep anti-bovine IgG (H+L) secondary antibody (Bethyl Lab. Montgomery, TX, USA). The precision protein Strep Tactin-HRP conjugate (Bio-Rad) was used as a secondary antibody for molecular weight markers. Finally, the membrane was washed and 25 mL of peroxidase substrate (TMB membrane HRP substrate (SeraCare Life Sciences Inc, Milford, MA, USA) was added, and the reaction was fully developed at room temperature. Protein band images were taken using a ChemiDoc™ Touch Imaging System (Bio-Rad) and analyzed using Image Lab Software (Bio-Rad). Images on the gel and the membrane were compared to identify immune-reactive protein bands.