Chondroitin sulphate b

To quantify the content of GAG in the samples, the reaction of GAG with dimethylmethylene (DMMB) blue was measured. This technique relies on the absorption spectra of the change in 1,9-dimethylmethylene dye due to the induction of metachromasia when it binds to sulfated GAGs.⁵¹ (link)
Frozen cartilage sections were digested overnight at 56 °C in 500 μl 1 mg/ml proteinase-K solution in digestion buffer (Tris/EDTA buffer). dimethylmethylene blue chloride (16 μg/ml dimethylmethylene blue) (Sigma-Aldrich, USA) in a solution of 3.04 mg/ml glycine and 2.37 mg/ml NaCl dissolved in 0.01M HCl in d₂H₂O (pH 3) was added directly followed by the measurement of the absorbance at 520 nm using a Varioskan Flash Multimode Reader (Thermo Scientific). Chondroitin sulphate-B (Sigma-Aldrich, USA) was used to generate a standard curve, and GAG measurements were normalized to the wet weight.⁵² (link) An ANOVA with a selected significance threshold of p < 0.05 was used to determine statistically significant differences between the GAG content for each layer of cartilage.

D-Glucuronic acid, chondroitin sulphate B, chondroitinase ABC, chondroitinase AC, Streptomyces hyaluronidase, β-D-galactosidase, sodium dodecyl sulphate, coomassie blue R-250, N,N-methylene bisacrylamide, and ß-mercaptoethanol were purchased from Sigma Chemical Company, St. Louis, USA. Pepsin, proteinase K, and potassium acetate were procured from Sisco Research Laboratory, Mumbai, India. All other reagents were of high analytical grade.

The sulphated sugar content of papain-digested samples (6 m HCl at 120 °C for 4 h) was determined using dimethylene blue (Sigma) according to Farndale et al. (1986 (link)). The GAG content was determined by interpolating values from a standard curve of chondroitin sulphate B (Sigma).