Biotinylated hyaluronan: A versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure

Hyaluronan influences cellular proliferation and migration in developing, regenerating and remodelling tissues and in tissues undergoing malignant tumour‐cell invasion. The widespread occurrence of hyaluronan‐binding proteins indicates that the recognition of hyaluronan is important to tissue organisation and the control of cellular behaviour. A number of extracellular matrix and cellular proteins, which have been termed the hyaladherins, have specific affinities for hyaluronan. These include cartilage link‐protein, hyaluronectin, neurocan, versican and aggrecan, which all bind to HA within the extracellular matrix. Cellular receptors for hyaluronan such as CD44 and RHAMM (receptor for hyaluronate‐mediated motility) have also been identified. In the present study biotinylated hyaluronan (bHA) was prepared by reacting adipic dihydrazide with a 170 kDa hyaluronan sample using the bifunctional reagent 1‐ethyl‐3‐[3‐(dimethylamino) propyl] carbodiimide. The resultant free amine moeity of the hydrazido‐hyaluronan was then reacted with biotin succinimidyl ester (sulfo‐NHS‐biotin) to prepare the bHA. After 4–20% gradient sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and electroblotting to nitrocellulose membranes, bHA and avidin alkaline phosphatase conjugate could be used in conjunction with nitroblue tetrazolium/5‐bromo‐4‐chloro‐3‐indolyl phosphate substrates to specifically visualise with high sensitivity (≥2 ng), bovine nasal cartilage link‐protein, aggrecan hyaluronan binding region, and human fibroblast hyaluronan receptors such as CD‐44. Conventional Western blotting using specific monoclonal antibodies to these proteins was also used to confirm the identities of these proteins.