Microassay for Sulfated Glycosaminoglycans by Alcian Blue Staining. Effects of Polyanionic Macromolecules on Staining in Untreated Biological Samples.

  • Yamamoto Eiji
    Department of Periodontology, Asahi University School of Dentistry
  • Kawabata Yoshikatsu
    Department of Periodontology, Asahi University School of Dentistry
  • Kou Yoshitada
    Department of Periodontology, Asahi University School of Dentistry
  • Kawai Kougorou
    Department of Periodontology, Asahi University School of Dentistry
  • Taniguchi Masato
    Department of Periodontology, Asahi University School of Dentistry
  • Takatori Tsukasa
    Department of Periodontology, Asahi University School of Dentistry
  • Imai Kenji
    Department of Periodontology, Asahi University School of Dentistry
  • Shiraki Masafumi
    Department of Periodontology, Asahi University School of Dentistry
  • Iwayama Yukio
    Department of Periodontology, Asahi University School of Dentistry

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Other Title
  • 硫酸化グリコサミノグリカンのアルシアンブルー染色による微量分析  生体粗試料直接測定時における陰性荷電高分子物質の影響
  • Effects of Polyanionic Macrom- olecules on Staining in Untreated Biological Samples
  • -生体粗試料直接測定時における陰性荷電高分子物質の影響-

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Abstract

Alcian blue has been used to detect glycosaminoglycans after they have been separated by electrophoresis on cellulose acetate membranes.<BR>We have already described a microprocedure that can be used for direct measurement of sulfated glycosaminoglycans by using an Alcian blue dyebinding assay without interferences by other anions such as DNA, hyaluronate, or bovine serum albumin.<BR>The aim of this study was to evaluate interference with direct measurement of sulfated glycosaminoglycans, by other anionic macromolecules in biological fluids.<BR>Biological samples, 2μl each, were blotted onto cellulose acetate strips and stained with 0.2% Alcian blue containing 0.05M MgCl2, 0.4M guanidine-HC1, 0.02M sulfuric acid, and 0.25% TritonX-100 at pH1.5. After drying at room temperature, the dye-substrate complex was analyzed with a dualwave length chromatoscanner. The amount of dye bound to anionic macromolecules in human saliva, serum, and synovial fluid was minimal. The sulfated glycosaminoglycan levels determined by direct assay were approximately 53% of the levels of purified gingival sulfated glycosaminoglycans measured by the electrophoretic method. This procedure is technically simple, rapid, and sensitive, and is useful even in the presence of other polyanions associated with biological fluids, although the yield of sulfated glycosaminoglycans must be improved.

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