Pa g column

To compare the internal structures of CS chains from the cartilage of different parts of Chinese sturgeon, 10 μg of each purified CS sample was partially digested at 37 °C for 24 h with 7.5 U of hyaluronidase from sheep testes. The digests were labeled with 2-AB and fractionated on a Superdex^TM Peptide 10/300 GL column eluted with 0.2 M NH₄HCO₃ at a flow rate of 0.4 mL/min for 60 min with fluorescent detection. The 2-AB-labeled oligosaccharide fractions were collected, freeze-dried, and analyzed by anion-exchange HPLC on a YMC-Pack PA-G column eluted with a linear gradient of NaH₂PO₄ at a flow rate of 1 mL/min at room temperature.

GAGs from splenic cell preparations were prepared as described previously^{23 (link)} with slight modifications. Briefly, cells were homogenized in ice-cold acetone at least thrice and air-dried. The dried materials (acetone powders) were exhaustively digested with heat-activated actinase E [10% (w/w) of dried materials] in 0.1 M borate buffer, pH 8.0, containing 10 mM CaCl₂, at 55 °C for 48 h. The digest was treated with 5% trichloroacetic acid, and the resultant acid-soluble fraction was adjusted to contain 80% ethanol. The resultant precipitate was dissolved in water and subjected to gel filtration on a PD-10 column (Cytiva, MA, USA) using water as an eluent. The flow-through fractions were collected, evaporated to dryness, and dissolved in water (crude GAG fractions).
An aliquot of the crude GAG sample was digested with a chondroitin sulfate-degrading enzyme, chondroitinase ABC (5 mIU, Seikagaku, Tokyo, Japan) in 50 mM Tris–HCl, and 60 mM sodium acetate, pH 8.0, at 37 °C for 2 h. The digests were derivatized with the fluorophore 2-aminobenzamide and were then analyzed by anion-exchange HPLC on a PA-G column (YMC, Kyoto, Japan) as described previously^{24 (link)}. Identification and quantification of the resulting disaccharides were achieved by comparison with authentic unsaturated CS disaccharides (Seikagaku).