Capillary blotting of glycosaminoglycans on nitrocellulose membranes after agarose-gel electrophoresis separation.
A method for the blotting and immobilizing of several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose gel electrophoresis is illustrated. This new approach to the study of glycosaminoglycans (GAGs) utilizes the capacity of agarose gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses.Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride and mixtures of GAGs are capillary blotted after their separation in agarose gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 microg. Nonsulfated polyanions, for example hyaluronic acid, may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 microg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes are used for immunological detection or other applications.
Volpi
Nicola
N
Department of Biologia Animale, University of Modena and Reggio Emilia, Via Campi 213/d, 41100, Modena, Italy. volpi@unimo.it
Maccari
Francesca
F
D002854:Chromatography, Paper / Q000295:instrumentation / Q000379:methods; D003101:Collodion / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation / Q000379:methods; D006025:Glycosaminoglycans / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods
D006025:Glycosaminoglycans; D003101:Collodion