Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling (1)
The functioning ability of cartilage is dependent on the maintenance of the extracellular matrix (ECM), which is an integrated assembly of collagens, glycoproteins, proteoglycans, and glycosaminoglycans (GAGs). Sulfated GAGs are linear polysaccharides that consist of repeating disaccharide units that are attached to proteoglycan core proteins on adherent animal cell surfaces and in extracellular matrices.
Explants of juvenile bovine shoulder and adult human knee cartilage were digested with papain (1% papain suspension, 5 mM cysteine, and 10 mM EDTA, pH 7.4) in a 60°C oven for 16 h. and the GAG content measured using a dimethyl-methylene blue (DMMB) assay (2).
1,9-Dimethylmethylene blue (DMMB) solutions (50 mg/l) was prepared by dissolving 50 mg of DMMB in 5 ml of ethanol and subsequent dilution to a volume of 1000 ml with 0.2% (w/v) sodium formate buffer, pH 3.5. 150ul of DMMB reagent was rapidly added to each well containing the digested cartilage sample. The plate was briefly mixed on a plate shaker (5–10 s) and the absorbance at 650 nm measured immediately in a plate reader.
