Exposure of binding sites for antibodies and concanavalin A on collagen by solubilization in hot urea. An immunoblot analysis.
The presence of urea during solubilization of collagenous samples for SDS PAGE had a marked effect upon mobility of collagenous polypeptides and upon binding of antibodies from certain rabbit antisera, antibodies from several human sera and binding of concanavalin A. When samples were solubilized with urea by heating at 100 degrees C the mobility of collagenous polypeptides was retarded relative to samples that had been heated without urea or exposed to urea without heating. Antibodies from the rabbit sera only bound on immunoblots to collagen that had been urea/heat-treated. Periodate oxidation and deglycosylation with trifluoromethanesulfonic acid abolished binding of the rabbit antibodies. The results indicate the presence of carbohydrate epitopes buried within collagenous polypeptides that are exposed by harsh denaturing conditions. Heating with urea appears to cause an unfolding of collagenous molecules beyond that produced by SDS solubilization without urea. These results underscore the necessity to pay close attention to conditions used to solubilize for electrophoresis samples that are subsequently used as targets for antibodies or other ligands.
D001665:Binding Sites; D003094:Collagen / Q000276:immunology* / Q000378:metabolism; D003166:Complement Activating Enzymes / Q000276:immunology; D015922:Complement C1q; D003208:Concanavalin A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D000939:Epitopes; D005109:Extracellular Matrix / Q000032:analysis; D006023:Glycoproteins / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D014508:Urea / Q000494:pharmacology*
D000939:Epitopes; D006023:Glycoproteins; D003208:Concanavalin A; D015922:Complement C1q; D014508:Urea; D003094:Collagen; D003166:Complement Activating Enzymes