Agren U M, Tammi R, Tammi M
Department of Anatomy, University of Kuopio, Finland.
Anal Biochem. 1994 Mar;217(2):311-5. doi: 10.1006/abio.1994.1124.
Glycosaminoglycans labeled in tissue culture in the presence of [3H]glucosamine were proteolytically solubilized and then precipitated with cetylpyridinium chloride onto a sheet of nitrocellulose using a dot-blot apparatus. The proportion of hyaluronan (HA) was calculated from parallel aliquots digested with Streptomyces hyaluronidase, an enzyme specifically degrading HA. A linear response of radioactivity was obtained for samples in the range of 50-10,000 cpm (1-1000 ng total HA) when the dots cut from the membrane were counted with liquid scintillation. Negligible interference from an excess of unincorporated precursor, chondroitin sulfate, and proteolytic tissue digest was observed. The assay was particularly convenient in measuring large numbers of samples with relatively low activity, such as testing the effects of different drug concentrations and analyzing chromatographic fractions.
在含有[3H]葡萄糖胺的组织培养中标记的糖胺聚糖经蛋白水解溶解，然后使用点杂交仪用十六烷基吡啶氯化物沉淀到硝酸纤维素膜上。透明质酸（HA）的比例由用特异性降解HA的酶——链霉菌透明质酸酶消化的平行等分试样计算得出。当用液体闪烁计数法对从膜上切下的点进行计数时，在50 - 10,000 cpm（1 - 1000 ng总HA）范围内的样品获得了放射性的线性响应。观察到过量未掺入的前体、硫酸软骨素和蛋白水解组织消化物的干扰可忽略不计。该测定法在测量大量活性相对较低的样品时特别方便，例如测试不同药物浓度的效果和分析色谱馏分。
