Hrp conjugated goat anti human fab specific antibody

Whey was separated on 12% SDS-PAGE gels under both reducing and non-reducing conditions. Anti-CD20 mAb was detected using an HRP-conjugated goat anti-human Fab-specific antibody (Sigma, St. Louis, MO) and ECL Western blot reagents (GE Healthcare). Purified human IgG (Sigma) was used as a positive control.

Milk and blood from two transgenic lines of mice and non-transgenic mice were collected at day 3 (colostrum) and day 10 (mature milk) of lactation. The milk was diluted with distilled water to 200 µl and defatted by centrifugation at 4°C for 10 min at 4,500×rpm. The serum was separated by centrifugation at 4°C for 5 min at 3,600×rpm and frozen at −80°C for future assay.
The skimmed milk and the serum obtained were mixed with sample buffer and then separated on 10% SDS-PAGE gels under reducing conditions. The proteins were visualized by staining with Coomassie brilliant blue (S4 Fig.). Separated proteins were electrophoretically transferred to nitrocellulose membranes (Amersham Pharmacia, UK), and then incubated overnight in blocking buffer (3% BSA in TBST) at 4°C. The immunodetection of human IgGs was performed with the HRP-conjugated goat anti-human Fab specific antibody (Sigma, USA), and purified human IgG (Sigma, USA) was used as the positive control. Besides, the HRP-conjugated anti-bFcRn mouse antibody (K6) was used for the bFcRn detection in the mammary gland. The images were developed with ECL western blotting reagents (Amersham Biosciences, UK) according to the manufacturer’s instructions. Protein containing the bFcRn was extracted from the mammary gland of cattle at early time of lactation and served as a positive control.