Lkb system

OPN was purified from human milk as described [27] (link). The purity of the protein was determined by N-terminal sequencing and SDS-PAGE. N- and C-terminal fragments of OPN were generated by digestion with thrombin (30 mU/µg OPN) in 0.1 M ammonium bicarbonate at 37°C for 1 h. The fragments were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Vydac C₁₈ column connected to a GE Healthcare LKB system. Separation was carried out in 0.1% trifluoroacetic acid (buffer A) and eluted with a gradient of 75% 2-propanol in 0.1% trifluoroacetic acid (buffer B) developed over 80 min (0–5 min, 0% buffer B; 5–70 min, 0–50% buffer B; 70–80 min, 50–95% buffer B) at a flow rate of 0.85 ml/min. The fragments were detected in the effluent by measuring the absorbance at 226 nm.

Bovine milk OPN (Lacprodan^® OPN-10) was obtained from Arla Foods Ingredients (Viby J, Denmark). Lacprodan^® OPN-10 contains ~78% protein, of which 95% is OPN. The full-length OPN and the N-terminal OPN fragments were separated by gel filtration on a Superdex 200 HR 10/30 column (GE Healthcare, Uppsala, Sweden) connected to a GE Healthcare LKB system as described [8 (link)]. Proteose peptone component 3 (PP3) was purified from bovine milk as described [21 (link)].
Polyclonal antibodies against OPN and PP3 were raised in rabbits by DAKO A/S (Glostrup, Denmark) as described [10 (link)]. The monoclonal antibody MAB193p that recognises an epitope located in the N-terminal part of OPN was from BBI Solutions (Portland, ME, USA). For biotinylation, antibodies were dialysed against 0.1 M sodium borate buffer, pH 8.8, overnight at 4 °C. One-sixth (v/v) of 10 mg/mL of N-hydroxysuccinimide biotin was added to the antibodies. After 4 h of incubation at room temperature, the reaction was stopped with 1 M of ammonium bicarbonate per 250 μg of N-hydroxysuccinimide biotin. Finally, the biotinylated antibodies were dialysed against phosphate-buffered saline (PBS) overnight at 4 °C as described [5 (link)].