Differential use of Alcian blue and toluidine blue dyes for the quantification and isolation of anionic glycoconjugates from cell cultures: application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes.
Alcian blue and toluidine blue dyes form complexes with anionic glycoconjugates (AG) such as proteoglycans (PG) and glycosaminoglycans (GAG). However, the Alcian blue-AG complexes do not readily dissociate, while the toluidine blue-AG complexes do so in salt solutions. This differential dissociation of the dye-AG complexes has been utilized in the analysis and isolation of radiolabeled AG elaborated by articular chondrocyte cultures incubated with the radiolabeled precursors of AG. For the rapid quantification of newly synthesized (35)S-labeled PG, small replicate aliquots of the radiolabeled culture media were applied directly to cellulose acetate strips, stained with Alcian blue and the stained immobilized radiolabeled PG was quantified by liquid scintillation counting. Comparison of anionic glycoconjugates quantified in the culture media employing toluidine blue and Alcian blue staining on cellulose acetate trips gave similar results. Staining on cellulose acetate strips using these two dyes is particularly suited for the simultaneous processing of large numbers of samples, as illustrated by the screening of the effects of biological materials and drugs on AG synthesis, in cultures labeled with [(35)S]-sulfate and [(3)H]-glucosamine. The Alcian blue and toluidine blue precipitation methods yielded similar results for the total AG recovered from the media of TGF-beta-stimulated chondrocytes. Electrophoretic analysis of toluidine blue- and Alcian blue-precipitated AG followed by autoradiography and Alcian blue staining in combination with silver nitrate demonstrated that both dyes yielded similar pattern of bands on gels. However, some AG from Alcian blue precipitate did not enter the gel, suggesting incomplete dissociation of Alcian blue-AG complex. The application of the toluidine blue precipitation method, in combination with enzymatic digestion of the GAG chains of the PGs, is illustrated by the isolation of a non-PG high-molecular-weight AG, as well as the PGs from the media of chondrocyte cultures stimulated by TGF-beta.
Terry
D E
DE
Department of Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Chopra
R K
RK
Ovenden
J
J
Anastassiades
T P
TP
D000423:Alcian Blue / Q000737:chemistry*; D000818:Animals; D000838:Anions; D002358:Cartilage, Articular / Q000166:cytology; D002417:Cattle; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004396:Coloring Agents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006001:Glycoconjugates / Q000032:analysis* / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D011509:Proteoglycans / Q000096:biosynthesis*; D013194:Staining and Labeling / Q000379:methods; D014048:Tolonium Chloride / Q000737:chemistry*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D000838:Anions; D004396:Coloring Agents; D006001:Glycoconjugates; D006023:Glycoproteins; D011509:Proteoglycans; D016212:Transforming Growth Factor beta; D014048:Tolonium Chloride; D000423:Alcian Blue