AN AGAROSE-GEL ELECTROPHORETIC METHOD FOR ANALYSIS OF HYALURONAN MOLECULAR-WEIGHT DISTRIBUTION
AN AGAROSE-GEL ELECTROPHORETIC METHOD FOR ANALYSIS OF HYALURONAN MOLECULAR-WEIGHT DISTRIBUTION
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DOI：
10.1006/abio.1994.1267
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发表时间：
1994-06-01
影响因子：
2.9
通讯作者：
COWMAN, MK
中科院分区：
文献类型：
--
作者：
LEE, HG;COWMAN, MK
描述了一种测定透明质酸（HA）分子量分布的电泳方法。该方法包括在0.5%琼脂糖凝胶上电泳分离透明质酸，然后使用阳离子染料Stains-All（3,3'-二甲基-9-甲基-4,5,4' -二苯并噻吩碳菁）检测透明质酸。推荐的样品负载为7 pg。用已知分子量的HA标准品校准该方法，在大约0.2-6 X 10的范围内建立了电泳迁移率与重量-平均分子量的对数之间的线性关系(6)。将透明质酸从琼脂糖凝胶电转移到尼龙膜后，分离的透明质酸图案也可以可视化。膜可染成阿利新蓝。或者，可以通过用生物素标记的HA结合蛋白探测尼龙膜，随后与链霉亲和素连接的金试剂相互作用并进行银染色扩增来实现对不纯样品的HA特异性检测。采用电泳法对两种不同液体结缔组织中的HA进行了分析。正常人膝关节滑液的HA分子量分布较窄，在6-7 X 10处达到峰值(6)。鸮猴玻璃体透明质酸的分子量分布也很窄，峰值在5-6 × 10处(6)。这些结果与现有公布的数据吻合良好，表明该方法适用于分析数量有限的不纯HA样品。(C) 1994学术出版社，Inc.
An electrophoretic method is described for determining the molecular weight distribution of hyaluronan (HA). The method involves separation of HA by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'-dibenzothiacarbocyanine). The recommended sample load is 7 pg. Calibration of the method with HA standards of known molecular weight has established a linear relationship between electrophoretic mobility and the logarithm of the weight-average molecular weight over the range of approximately 0.2-6 X 10(6). The separated HA pattern may also be visualized after electrotransfer of HA from the agarose gel to a nylon membrane. The membrane may be stained with the dye alcian blue. Alternatively, specific detection of HA from impure samples can be achieved by probing the nylon membrane with biotin-labeled HA-binding protein and subsequent interaction with a streptavidin-linked gold reagent and silver staining for amplification. The electrophoretic method was used to analyze HA in two different liquid connective tissues. Normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 X 10(6). Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 X 10(6). These results agi ee well with available published data and indicate the applicability of the method to the analysis of impure HA samples which may be available in limited amounts. (C) 1994 Academic Press, Inc.