Buee L, Boyle N J, Zhang L B, Delacourte A, Fillit H M
Ritter Department of Geriatrics, Mount Sinai Medical Center, New York, New York 10029.
Anal Biochem. 1991 Jun;195(2):238-42. doi: 10.1016/0003-2697(91)90323-l.
A modification of a method for the detection of proteoglycans on positively charged nylon was adapted for use in a dot-blot assay and improved. Different dot-blot membranes were tested and variations in the Alcian blue staining solution (including pH, critical electrolyte concentration, and ionic strength) were explored. With modifications, we were able to eliminate interferences by other polyanions such as DNA or proteins. We were able to detect glycosaminoglycans and proteoglycans down to the 10 ng range. Furthermore, our assay is compatible with high concentration of urea (up to 7 M) used in classic proteoglycans extraction methods and is a useful tool to monitor the isolation of proteoglycans by anion exchange and gel filtration chromatography. It is technically easier, faster, more sensitive, and more specific than previously published methods and can be adaptated as a quantitative assay using a scanning densitometer with a linear range of detection from 10 to 100 ng of glycosaminoglycans.
一种用于检测带正电荷尼龙上蛋白聚糖的方法经过改良后用于斑点印迹分析并得到了改进。测试了不同的斑点印迹膜，并探索了阿尔新蓝染色液的变化（包括pH值、临界电解质浓度和离子强度）。通过改良，我们能够消除DNA或蛋白质等其他聚阴离子的干扰。我们能够检测低至10 ng范围内的糖胺聚糖和蛋白聚糖。此外，我们的分析方法与经典蛋白聚糖提取方法中使用的高浓度尿素（高达7 M）兼容，是监测通过阴离子交换和凝胶过滤色谱法分离蛋白聚糖的有用工具。它在技术上比以前发表的方法更简便、更快、更灵敏、更特异，并且可以使用扫描密度计改编为定量分析方法，其检测线性范围为10至100 ng糖胺聚糖。
