Quantification of 35S-labeled proteoglycans complexed to alcian blue by rapid filtration in multiwell plates.
This paper describes a rapid filtration assay for the quantification of 35S-labeled proteoglycans and/or 35S-labeled glycosaminoglycans in a large number of samples. Separation of 35S-labeled proteoglycans and 35S-labeled glycosaminoglycans from unincorporated [35S]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 microns pore size) fitted in a 96-well plate. Following brief rinsing steps, the discs are punched out and 35S-labeled macromolecules retained on the membrane are then quantified by scintillation counting. In this rapid filtration assay, the relationship between the amount of [35S]-aggrecan applied and radioactivity measured was linear over a broad range of concentrations (2-800 micrograms aggrecan/ml). The amount of 35S-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 micrograms/ml), serum (up to 30%), or guanidine hydrochloride at 4 M did not affect recovery of 35S-labeled proteoglycans measurably. CPM values obtained for 35S-labeled proteoglycans or 35S-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.
Masuda
K
K
Department of Biochemistry, Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612.
Shirota
H
H
Thonar
E J
EJ
D000423:Alcian Blue; D000818:Animals; D002358:Cartilage, Articular / Q000737:chemistry / Q000166:cytology; D002417:Cattle; D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods; D004247:DNA / Q000494:pharmacology; D003911:Dextrans; D005374:Filtration / Q000379:methods; D006025:Glycosaminoglycans / Q000032:analysis; D019791:Guanidine; D006146:Guanidines; D008297:Male; D008567:Membranes, Artificial; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology; D011509:Proteoglycans / Q000032:analysis*; D013462:Sulfur Radioisotopes
D003911:Dextrans; D006025:Glycosaminoglycans; D006146:Guanidines; D008567:Membranes, Artificial; D011506:Proteins; D011509:Proteoglycans; D013462:Sulfur Radioisotopes; D004247:DNA; C025614:sephadex; D019791:Guanidine; D000423:Alcian Blue