A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver.
A sensitive method has been developed for the visualization of nonradiolabeled glycosaminoglycan oligosaccharides resolved by polyacrylamide gel electrophoresis using fixation with azure A followed by staining with ammoniacal silver. This method, which can detect as little as 1-2 ng of a single oligosaccharide species, can be used to stain a few micrograms of a complex oligosaccharide mixture. The combination of gradient polyacrylamide gel electrophoresis and sequential azure A/silver staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate) and hyaluronate, as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited and/or where radiolabeling is impractical or undesirable.
Cancer Research Campaign Department of Medical Oncology, Christie Hospital, Manchester, United Kingdom.
M
M
Lyon
J T
JT
Gallagher
D001399:Azure Stains; D002809:Chondroitin Sulfates / Q000032:analysis; D003871:Dermatan Sulfate / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006025:Glycosaminoglycans / Q000032:analysis*; D006493:Heparin / Q000032:analysis; D006497:Heparan Sulfate / Q000378:metabolism; D007632:Keratan Sulfate / Q000032:analysis; D009844:Oligosaccharides / Q000032:analysis*; D010316:Particle Size; D010640:Phenothiazines; D011133:Polysaccharide-Lyases / Q000378:metabolism; D012834:Silver; D013194:Staining and Labeling; D013379:Substrate Specificity
D001399:Azure Stains; D002809:Chondroitin Sulfates; D003871:Dermatan Sulfate; D006025:Glycosaminoglycans; D006493:Heparin; D006497:Heparan Sulfate; D007632:Keratan Sulfate; D009844:Oligosaccharides; D010640:Phenothiazines; D011133:Polysaccharide-Lyases; D012834:Silver; C000212:ammoniacal silver; C025818:Azure A