*13.2. Aggrecan Isolation Procedures*

To isolate aggrecan from cartilage for analysis, it must be dissociated from its ternary complex formation with HA and link protein. This is achieved by using chaotropic agents such as guanidinium hydrochloride (GuHCl), which disrupts the water structure of the tissue, opens up the dense collagenous structure allowing dissociation of the aggrecan–HA–link protein complexes and release of aggrecan monomer which diffuses out of the tissue and is recovered in the extraction solution [228]. Cartilage is initially diced into small pieces to reduce the diffusive pathways out of the tissue for effective extraction; broad spectrum protease inhibitors covering all four mechanistic classes of proteases are included in the extraction solution to protect the aggrecan from proteolysis. Homogenisation procedures, which are commonly used in the extraction of proteins from other soft connective tissues, cannot be used for the isolation of aggrecan in an intact form since their high shear forces fragment the aggrecan. The cartilage extract can then be subjected to anion exchange chromatography on support matrices derivatised with anionic ligands such as diethylaminoethyl (DEAE) or sulphopropyl [229], dissociative size exclusion chromatography in 4-M GuHCl containing buffers using open pore gel chromatographic media such as Sephacryl or Sepharose CL2B [230], or density gradient equilibrium isopycnic ultracentrifugation in high concentrations of CsCl [231]. This latter procedure relies on aggrecan's high buoyant density in CsCl gradients of ≥1.55 g/mL to isolate aggrecan; while extracted proteins typically have buoyant densities of 1.3–1.35 g/mL, HA has a buoyant density of 1.4–1.45 g/mL. Density gradient ultracentrifugation can be conducted in the presence of 4M GuHCl to ensure aggrecan is isolated free of other interactive components also present in the cartilage extract [231,232].
