A quantitative solid-phase assay for identifying radiolabeled glycosaminoglycans in crude cell extracts.
Extraction of radiosulfate-labeled cell layers in denaturing urea and nonionic detergent allows the quantitative binding of GAG-containing materials from up to 96 discrete samples to a single cationic nylon blot. Free sulfate and/or sulfated lipids fail to bind. Washing the blot with differential salt concentrations discriminates between native proteoglycans and free glycosaminoglycan chains or fragments. In addition, chondroitin sulfates and heparan sulfate are identified either by prior digestion with chondroitin ABC or AC lyase, as generated disaccharides fail to bind to the blot, or by treatment of the entire blot with nitrous acid following binding. Similarly, heparan sulfate can be identified on chromatograms or Western transfers from polyacrylamide gel electrophoresis by autoradiography before and after treatment of the blot with nitrous acid.
Rapraeger
A
A
Department of Pathology, University of Wisconsin, Madison 53706.
Yeaman
C
C
D000818:Animals; D001665:Binding Sites; D002809:Chondroitin Sulfates / Q000032:analysis; D002853:Chromatography, Liquid; D006025:Glycosaminoglycans / Q000032:analysis*; D006497:Heparan Sulfate / Q000032:analysis; D008567:Membranes, Artificial; D051379:Mice; D009757:Nylons; D014020:Tissue Extracts / Q000032:analysis*; D014407:Tumor Cells, Cultured
D002809:Chondroitin Sulfates; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D008567:Membranes, Artificial; D009757:Nylons; D014020:Tissue Extracts