Arabinan came from sugar beet (product code [PC] P-ARAB), pectic galactan was obtained from potato (PC P-PGAPT), polygalacturonic acid was from citrus pectin (PC P-PGACT), and rhamnogalacturonan I was from potato (PC P-RHAM1); these glycans were all purchased from Megazyme International (Wicklow, Ireland). Amylopectin from maize (PC 10120) and chondroitin sulfate from bovine trachea (PC C9819) were purchased from Sigma-Aldrich (St. Louis, MO). The chemical structures of the glycans are illustrated in Fig. 1 . Stocks for each glycan (10 mg/ml) were prepared using purified water and sterilized by autoclaving for 20 min at 121°C.
Chondroitin sulfate from bovine trachea
Manufactured by Merck Group
Sourced in United States
Chondroitin sulfate from bovine trachea is a natural compound extracted from the cartilage of bovine trachea. It is a glycosaminoglycan that is a primary structural component of cartilage and other connective tissues. The product is suitable for use in various research and analytical applications.
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Lab products found in correlation
4 protocols using chondroitin sulfate from bovine trachea
1
Characterization of Glycan Stocks
2
Quantifying Sulfated Glycosaminoglycan Production
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On day 6, one half of each agarose construct was used to examine for PG content. Each sample was placed in 500 µL papain buffer (pH 7.4) containing 250 µg/ml papain (Sigma-Aldrich, St. Louis, Mo), 100 mM sodium phosphate, 10 mM EDTA and 10 mM cysteine HCl and incubated at 60 °C for 16 h. The amount of sulfated glycosaminoglycan in the digestion solution was determined by Dimethylmethylene (DMMB) blue dye-binding assay. Briefly, 20 µL aliquot of the papain digest was mixed with 200 µL of DMMB staining solution (pH = 1.5)85 . Absorbance of the mixture was read at 525 nm by the multimode plate reader. Chondroitin sulfate from bovine trachea (Sigma-Aldrich, St. Louis, Mo) dissolved in the same papain buffer was used as the standard. All measurements were normalized by DNA content as determined by the Hoechst 33258 fluorometric assay86 (link). The PG production was defined as an increased amount of PG content over 6 days by comparing with the PG content of agarose constructs on day 0.
3
Quantifying GAG Content in Cell Pellets
4
Proteolytic Digestion of Glycosaminoglycans
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All chemicals and solvents were of analytical grade and used as received from commercial sources. Alcalase ® 2.41 serine-protease from Bacillus licheniformis was purchased from Novozymes ® (Bagsvaerd, Denmark). GAGs standard, heparan sulfate from bovine kidney, chondroitin sulfate from bovine trachea and dermatan sulfate from porcine intestinal mucosa
were from Sigma-Aldrich (St. Louis, MO, USA). Chondroitinase ABC, chondroitin ABC lyase, from Proteus vulgaris (EC 4.2.2.4), specific activity of 0.5-2 units/mg, and chondroitinase AC, chondroitin AC lyase, from Flavobacterium heparinum (EC 4.2.2.5), specific activity of 0.5-1.5 units/mg, were from Sigma-Aldrich. Unsaturated chondro/dermato disaccharides [ΔDi0S (ΔUA-
and ΔDi2,4,6triS (ΔDitriS, ΔUA-2S-[1→3]-GalNAc-4S,6S)] were from Seikagaku Corporation (Tokyo City, Japan) and Sigma-Aldrich. Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'dibenzothiacarbocyanine) was from Sigma-Aldrich. QAE Sephadex ® A-25 anion-exchange resin was from Pharmacia Biotech (Uppsala, Sweden). All other chemicals and reagents used were of analytical grade.
were from Sigma-Aldrich (St. Louis, MO, USA). Chondroitinase ABC, chondroitin ABC lyase, from Proteus vulgaris (EC 4.2.2.4), specific activity of 0.5-2 units/mg, and chondroitinase AC, chondroitin AC lyase, from Flavobacterium heparinum (EC 4.2.2.5), specific activity of 0.5-1.5 units/mg, were from Sigma-Aldrich. Unsaturated chondro/dermato disaccharides [ΔDi0S (ΔUA-
and ΔDi2,4,6triS (ΔDitriS, ΔUA-2S-[1→3]-GalNAc-4S,6S)] were from Seikagaku Corporation (Tokyo City, Japan) and Sigma-Aldrich. Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'dibenzothiacarbocyanine) was from Sigma-Aldrich. QAE Sephadex ® A-25 anion-exchange resin was from Pharmacia Biotech (Uppsala, Sweden). All other chemicals and reagents used were of analytical grade.
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