QUANTIFICATION OF S-35 LABELED PROTEOGLYCANS COMPLEXED TO ALCIAN BLUE BY RAPID FILTRATION IN MULTIWELL PLATES

This paper describes a rapid filtration assay for the quantification of S-35-labeled proteoglycans and/or S-35-labeled glycosaminoglycans in a large number of samples. Separation of S-35-labeled proteoglycans and S-35-labeled glycosaminoglycans from unincorporated [S-35]sulfate is effected by forming insoluble complexes between alcian blue and the glycosaminoglycan moieties of the proteoglycans and then filtering the solutions through ''Durapore membrane'' discs (0.45 mu m pore size) fitted in a 96-well plate. Following brief rinsing steps, the discs are punched out and S-35-labeled macromolecules retained on the membrane are then quantified by scintillation counting. In this rapid filtration assay, the relationship between the amount of [S-35]-aggrecan applied and radioactivity measured was linear over a broad range of concentrations (2-800 mu g aggrecan/ml). The amount of S-35-labeled proteoglycans measured in media and 4 M guanidine HCl extracts of articular cartilage and three different chondrocyte culture systems (monolayer, agarose gel, and alginate bead) ranged between 90 and 101% of the value obtained by sieve chromatography on Sephadex G-25. The presence in samples of unlabeled proteoglycans (up to 1 mg/ml), bovine serum albumin (up to 4 mg/ml), DNA (up to 20 mu g/ml), serum (up to 30%), or guanidine hydrochloride at 4 M did not affect recovery of S-35-labeled proteoglycans measurably. CPM values obtained for S-35-labeled proteoglycans or S-35-labeled glycosaminoglycans quantified by chromatography on Sephadex G-25 and the filtration assay showed a strong linear relationship (r > 0.99) irrespective of the type of culture medium, extract, or digest used. (C) 1994 Academic Press, Inc.