There are several sample preparation protocols for glycosaminoglycan (GAG) analysis, such as guanidine hydrochloride (GuCl) extraction, guanidine thiocyanate (GITC) extraction, and enzymatic digestion using papain or pepsin. Hoemann et al. confirmed that the papain digestion protocol is more effective than the GuCl or GITC extraction protocols for analyzing GAG, DNA, and collagen content in cartilage and repair tissues.
Glycosaminoglycan (GAG) Analysis:
The GAG content detected in papain-digested samples from hydrogel cultures or cartilage samples was similar to the content obtained using a 4 M GuCl extract. Using the developed multivalent extraction method, a small amount of additional GAG was detected in the guanidine-insoluble pellet after papain digestion, indicating that some GAG remained insoluble after guanidine extraction. Combining the guanidine-soluble and guanidine-insoluble fractions yielded GAG values similar to those obtained from samples directly digested with papain.
DNA Analysis:
Compared to direct papain digestion, the initial GuCl extract resulted in incomplete DNA recovery. Most of the DNA was recovered from the papain-digested or guanidine-insoluble pellet. Combining the GuCl extract with the papain digest of the insoluble material yielded over 90% of the total DNA content from cartilage or hydrogel-cultured chondrocytes, comparable to the yield obtained through direct papain digestion.
Total Collagen Analysis:
The collagen content was extrapolated from the hydroxyproline (HP) content using a HP assay. Papain digestion at 60°C denatures collagen and effectively extracts the denatured collagen by degrading into small peptides.
Highly cross-linked collagen was expected to remain insoluble after guanidine extractions. Thus, the final GITC-insoluble pellet was digested with papain and analyzed using the HP assay to determine the amount of insoluble collagen. Matched hydrogel and cartilage samples were subjected to direct papain digestion to determine total collagen content. In cultured hydrogels and cartilage samples, the amount of guanidine-insoluble collagen were around 50% and 80%, respectively, of the total collagen detected in corresponding the directly digested samples.
In summary, directly digesting samples with papain can provide a more accurate measurement of GAG, DNA, and collagen.
Chondrex Inc. offers assay kits for GAG (Cat# 6022), DNA (Cat# 6023), and total collagen (Cat# 6017) as well as collagen for scaffolds (Cat# 1202 and 1203), and a non-scaffold 3D culture system, TASCL (Cat# CTASCL6WELL) for your studies. For analysis of native collagen along with GAGs, please contact support@chondrex.com to discuss Chondrex Inc’s protocols.
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