Pa 03 column

CS from MC3T3-E1 and BMSC cultures was prepared as described previously42 (link). The purified glycosaminoglycan fraction containing CS was digested with chondroitinase ABC at 37°C for 2 h. The digests were derivatized with the fluorophore 2-aminobenzamide and were then analyzed via anion-exchange HPLC on a PA-03 column (YMC, Kyoto, Japan). Identification and quantification of the resulting disaccharides were achieved by comparison with authentic unsaturated CS disaccharides (Seikagaku).

The purified recombinant aggrecan-Fc was subjected to gel filtration on a PD-10 column (GE Healthcare). The flow-through fractions were collected and digested with 5 mIU of chABC (Seikagaku Corporation) in 60 mM sodium acetate, 50 mM Tris-HCl (pH 8.0) for 12 h at 37°C. The digests were derivatized with a fluorophore, 2-aminobenzamide, and then analyzed by anion-exchange high-performance liquid chromatography (SLC-10A, Shimadzu) on a PA-03 column (YMC). Identification and quantification of the resulting disaccharides were achieved by comparison with chondroitin sulfate-derived authentic unsaturated disaccharides (Seikagaku Corporation), as described previously [38 (link)].