Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan.
Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan.
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DOI：
10.1016/j.ab.2011.05.026
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发表时间：
2011-10-01
影响因子：
2.9
通讯作者：
Cowman MK
中科院分区：
文献类型：
--
作者：
Bhilocha S;Amin R;Pandya M;Yuan H;Tank M;LoBello J;Shytuhina A;Wang W;Wisniewski HG;de la Motte C;Cowman MK
研究了琼脂糖凝胶和聚丙烯酰胺凝胶电泳法在约5-500 kDa范围内对透明质酸(HA)的分子质量依赖性分离。对于琼脂糖基系统，确定了不同琼脂糖类、琼脂糖浓度和缓冲体系的适用性。利用化学酶法合成的低多分散度的HA标准，确定了各凝胶组成的分子质量范围，在该范围内，HA的迁移率与分子质量的对数呈线性关系。在琼脂糖凝胶中对低至约9 kDa的HA分子质量进行了良好的线性校正。对于更高分辨率的分离，以及延伸到低至约5 kDa的分子质量，梯度聚丙烯酰胺凝胶是更好的选择。染色凝胶的密度扫描可以分析样品中存在的分子质量范围，并计算重量平均值和数平均值。对于粘均分子质量分别为112、59、37和22 kDa的多分散HA样品，在0.5微克(对于聚丙烯酰胺)到2.5微克(对于琼脂糖)的样品负载下，该方法得到了验证。使用凝胶迁移率改变分析的方法，将聚集素的HA结合区(重组人聚集素G1-IGD-G2结构域)与150 kDa的HA标准结合。
Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition, over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample, and calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa, at sample loads of 0.5 µg (for polyacrylamide) to 2.5 µg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150 kDa HA standard.
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影响因子：
7
作者：
Durigova, M.;Roughley, P. J.;Mort, J. S.
通讯作者：
Mort, J. S.
影响因子：
3.8
作者：
EDENS, RE;ALHAKIM, A;LINHARDT, RJ
通讯作者：
LINHARDT, RJ
影响因子：
--
作者：
BALAZS, EA;WATSON, D;ROSEMAN, S
通讯作者：
ROSEMAN, S
DOI：
10.1016/0885-4505(86)90077-0
发表时间：
1986-04-01
期刊：
BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY
影响因子：
--
作者：
DAHL, LB;DAHL, IMS;BORRESEN, AL
通讯作者：
BORRESEN, AL
影响因子：
2.9
作者：
KNUDSON, W;GUNDLACH, MW;CONRAD, HE
通讯作者：
CONRAD, HE