Pack pa g column

Isolation and purification of GAG chains from the cell cultures were performed^{67 (link)}. Cells were homogenized with ice-cold acetone, extracted with ice-cold acetone three times, and air-dried thoroughly. The delipidated acetone powder was digested with actinase E (one-tenth the weight of acetone powder) in 0.1 M borate-sodium, pH 8.0, containing 10 mM CaCl₂ at 55 °C for 48 h. The samples were adjusted to 5% v/v in trichloroacetic acid and centrifuged. The GAG-containing materials were precipitated from the resultant supernatants by mixing with ethanol, dissolved in water, and subjected to gel filtration on a PD-10 desalting column (Cytiva) using water as an eluent. The flow-through fractions were collected and evaporated to dryness. The purified GAG fraction containing the CS and HS chains was digested with 5 mIU of chondroitinase ABC (Seikagaku), or a mixture of 0.5 mIU of heparinase (Seikagaku) and 0.5 mIU of heparitinase (Seikagaku), at 37 °C for 3 h. The digests were derivatized with the fluorophore 2-AB and then analyzed by anion-exchange HPLC using a YMC-Pack PA-G column (4.6 × 250 mm, YMC, Kyoto, Japan). A modular HPLC system (Shimadzu) was operated by LabSolutions LC/GC (Ver. 5.42, Shimadzu). The identification and quantification of the resulting disaccharides were achieved by comparison with authentic unsaturated CS and HS disaccharides (Seikagaku).

Disaccharide compositions of various CS/DS were analyzed by high HPLC as described previously (27 (link)). Briefly, CS/DS polysaccharides or oligosaccharides were individually digested by CSase ABC followed by 2-AB labeling and analyzed by anion exchange HPLC on a YMC Pack PA-G column (YMC-Pack) eluted with a linear gradient from 16 mM to 460 mM NaH₂PO₄ over 60 min at a flow rate of 1.0 ml/min at room temperature using a fluorescence detector with excitation and emission wavelengths of 330 and 420 nm, respectively. Identification and quantification of the resulting disaccharides were achieved by comparison with the elution positions of CS-derived authentic unsaturated disaccharides.