After cooling, the cooled extract was centrifuged (5000 × g for 10 min) and then filtered through a Whatman no.5 filter paper. The extract was stored at −20 °C until analysed. The residual tissue was further digested with papain, and uronic acid contents in both the extract and the residual tissue were determined by the carbazole Baf-A1 in vivo reaction (see Section 2.7). These estimates enabled the proportion of uronic acid liberated to be expressed as a percentage of the total uronic acid recovered. The total extractability of uronic acid was then compared between the different extraction conditions. The preparation of each extract, which was referred to as antler papain extract, was performed in triplicate and the entire

experiment was independently replicated three times to address precision. Antler CS fractions were isolated and examined for molecular size using Sephacryl S-300 chromatography (Pharmacia Biotech Inc., Quebec, Canada). A portion of the antler papain extract was fractionated using a 1 × 110 cm column equilibrated and eluted with 0.05 M NaCl buffer, pH 5.8, at a flow rate of 3 mL/h. Blue dextran IDO inhibitor and tritiated water were used to determine void volume (Vo) and total volume (Vt) of the column, respectively. The partition coefficient was calculated from the formula: Kav = (Ve−Vo)/(Vt−Vo), in which Ve represents the volume of the peak fraction. The eluates (1 mL) were analysed for protein at 280 nm absorbance, hydroxyproline,

sulfated GAG and uronic acid content as explained in Section 2.7. Antler CS fractions were pooled MTMR9 and freeze-dried for further study. All chromatography data presented in this paper are means of 3 experiments. Electrophoresis was performed in 0.6% acrylamide in agarose in Tris buffer, pH 6.8. Samples were dissolved in deionised water. Two slabs were

generally run at the same time, one for staining with toluidine blue and the other for western blot with monoclonal antibodies to chondroitin sulfate (CS-56) (Sigma–Aldrich, USA). Electrophoretic transfer to nitrocellulose was accomplished in Tris–borate (gel electrophoresis buffer) without sodium dodecyl sulfate at 40 V for 2 h. Nitrocellulose sheets were then soaked in 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), pH 7.2 for 1 h at room temperature. After washing in PBS (three times for 5 min each), nitrocellulose sheets were incubated with anti-CS monoclonal antibodies (in PBS containing 1% BSA) for 1 h at room temperature. The incubation was followed by washing in PBS and 1 h incubation with rabbit anti-mouse IgM conjugated with horseradish peroxidase. Colour was developed by incubating in 0.05% diaminobenzidine tetra-hydrochloride in PBS containing hydrogen peroxide (0.01% w/v) and cobalt chloride (0.033% w/v) for 5 min. Stained blots were then washed several times in water and dried. Hyaluronic acid from human umbilical cord (Sigma–Aldrich, USA) was dissolved in 0.5 M sodium acetate buffer, pH 6.