Polyacrylamide gel electrophoresis of fluorophore-labeled hyaluronan and chondroitin sulfate disaccharides: application to the analysis in cells and tissues.
This report describes a new formulation of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides (PAGEFS) for the analysis of hyaluronan (HA) and chondroitin sulfate (CS) Delta-disaccharides. PAGEFS relies on derivatization of reducing ends of HA- and the variously sulfated CS-derived Delta-disaccharides with 2-aminoacridone (AMAC), followed by electrophoresis under optimized buffer conditions (Tris-borate and Tris-HCl) and on polyacrylamide gels (25% T/3.75% C). The method was applied to the analysis of glycosaminoglycans (GAGs) from the human umbilical cord tissue and GAGs isolated from human aortic smooth muscle cell cultures. The obtained results were in agreement with those obtained after an analysis with high-performance liquid chromatography (HPLC). On the basis of these results, PAGEFS is a rapid and sensitive method for the analysis of the total amount of HA- and CS-derived disaccharides, as it allows analyzing 20 samples in minigels in one run and provides quantitation with relatively high sensitivity (less than 25 pmol per disaccharide). In addition, PAGEFS overcomes the lack of commercial gels described previously for the separation of AMAC-labeled disaccharides. Therefore, the method proposed here is an economic and useful tool for a fast screening of GAGs in biological samples, particularly when a high number of samples should be analyzed.
Karousou
Evgenia G
EG
Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Varese, Italy.
Militsopoulou
Maria
M
Porta
Giovanni
G
De Luca
Giancarlo
G
Hascall
Vincent C
VC
Passi
Alberto
A
D001011:Aorta; D002809:Chondroitin Sulfates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005459:Fluorides; D006025:Glycosaminoglycans / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry*; D007202:Indicators and Reagents; D009131:Muscle, Smooth, Vascular
D004187:Disaccharides; D006025:Glycosaminoglycans; D007202:Indicators and Reagents; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D005459:Fluorides