Development of a highly sensitive chemical method for detecting alpha2-->8-linked oligo/polysialic acid residues in glycoproteins blotted on the membrane.
A highly sensitive chemical method for detecting alpha2-->8-linked oligo/polysialic acid (oligo/polySia) chains was developed, including (i) periodate oxidation, reduction with sodium borohydride, and subsequent acid hydrolysis, giving rise to C7 analogues and intact C9 compounds from nonreducing terminal and internal sialic acid residues, respectively; (ii) fluorescent labeling of these C7 and C9 compounds with 1,2-diamino-4,5-methylenedioxybenzene (DMB); and (iii) quantitation of theseDMB derivatives on fluorometric high-performance liquid chromatography. As little as 1 ng of internal sialic acid residues of oligo/polySia chains, the existence of which indicates the presence of oligo/polySia structure, was detectable by this method. This fluorometric C7/C9 analysis was successfully applied to glycoproteins blotted onto a slit of polyvinylidene fluoride membranes and suggested the presence of some novel oligoSia-containing glycoproteins in pig embryonic brains.
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
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Sato
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Inoue
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Matsuda
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Kitajima
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000196:embryology; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000737:chemistry*; D010655:Phenylenediamines; D011145:Polyvinyls; D012680:Sensitivity and Specificity; D012794:Sialic Acids / Q000032:analysis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013552:Swine; D005465:Fluorocarbon Polymers
D005456:Fluorescent Dyes; D006023:Glycoproteins; D010655:Phenylenediamines; D011145:Polyvinyls; D012794:Sialic Acids; C021319:polysialic acid; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C058886:1,2-diamino-4,5-methylenedioxybenzene