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Quantification and characterization of glycosaminoglycans at the nanogram level by a combined azure A-silver staining
C H van de Lest1, E M Versteeg, J H Veerkamp
1Department of Biochemistry, University of Nijmegen, The Netherlands.
Abstract:
A rapid, sensitive, and nonradioactive method has been developed for the quantification and characterization of glycosaminoglycans. The method is based on the separation of different types of glycosaminoglycans in agarose gel and subsequent fixation and staining with the cationic dye azure A, followed by silver enhancement. Densitometric analysis of the silver deposition gives a linear response between 1 and 20 ng for chondroitin and dermatan sulfate and between 2 and 40 ng for heparan sulfate. The detection limit is about 250 pg. The staining procedure was applied for the quantification and characterization of glycosaminoglycans in human serum, urine, and lung and in mouse kidney glomeruli. It requires only 10 microliters serum, 2 microliters urine, and only a single cryosection in case of tissue. The method is at least as sensitive as staining with radioactive markers and about 200 times more sensitive than conventional glycosaminoglycan-staining methods.