Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains-All.
A sensitive method has been developed for the visualization of nonradiolabelled glycosaminoglycans resolved by agarose gel electrophoresis using staining with toluidine blue followed by Stains-All procedure. This method, which can detect as little as 10 ng of a single species, can be used to stain a few micrograms of a complex polysaccharide mixture. The combination of agarose gel electrophoresis and sequential toluidine blue/Stains-All staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate) and nonsulfated polyanions (i.e., hyaluronate, defructosylated capsular polysaccharide K4) as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes and the identification and quantification of the contaminations of other polysaccharides within glycosaminoglycan preparations with great sensitivity (about 0.1%). Furthermore, this method can be used to stain low-molecular-mass fractions and oligosaccharides derived from the natural polyanions, such as heparin. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited.
Volpi
Nicola
N
Department of Animal Biology, University of Modena and Reggio Emilia, Modena, Italy. volpi@unimo.it
Maccari
Francesca
F
D000818:Animals; D002232:Carbocyanines; D002356:Cartilage / Q000737:chemistry; D002417:Cattle; D004396:Coloring Agents; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004926:Escherichia coli; D006025:Glycosaminoglycans / Q000032:analysis / Q000302:isolation & purification*; D007202:Indicators and Reagents; D012680:Sensitivity and Specificity; D012754:Sharks; D014048:Tolonium Chloride
D002232:Carbocyanines; D004396:Coloring Agents; D006025:Glycosaminoglycans; D007202:Indicators and Reagents; D014048:Tolonium Chloride; C012040:stains-all