Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform
Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform
复制标题
整体固定化pH梯度毛细管等电聚焦柱的制备及其基于在线毛细管等电聚焦平台的蛋白质分离应用
DOI：
10.1002/elps.201900125
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发表时间：
2019-07-01
期刊：
影响因子：
2.9
通讯作者：
Yan, Chao
中科院分区：
文献类型：
--
作者：
Liu, Rangdong;Pan, Hong;Yan, Chao
以甲醇和正癸醇为致孔剂，通过甲基丙烯酸缩水甘油酯和聚乙二醇二丙烯酸酯的原位反应制备了部分填充毛细管整体柱。然后，将Pharmalyte 3-10固定在该柱上，以获得具有整体固定化pH梯度（M-IPG）的毛细管等电聚焦（cIEF）柱。此外，通过引入一个十字形单元和两个短路阀，建立了一个在线自建的蛋白质分离平台。在该平台中，十字形单元不仅用于连接M-IPG柱和六通进样阀（1.5 μ L样品环），而且还用于提供阳极缓冲液的体积池，以便可以顺序执行样品的进样、聚焦和移动过程。T形单元或十字形单元中的短路阀可用于控制流体流动的方向。利用该在线cIEF平台，在优化的条件下，可将7种蛋白质混合物分离，并在M-IPG柱上获得了pI值与迁移时间之间良好的线性相关性。同时，基于在线cIEF平台，新开发的M-IPG柱成功地分离了人血清蛋白和Hb A和Hb A（1c）的混合物。
In the presence of methanol and n-decanol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly (ethylene glycol) diacrylate. Then, Pharmalyte 3-10 was immobilized on this column in order to obtain a capillary isoelectric focusing (cIEF) column with monolithic immobilized pH gradient (M-IPG). In addition, an online self-built platform for protein separation was established on account of the introduction of a cross-shaped unit and two short-off valves. In this platform, a cross-shaped unit was not only used to join the M-IPG column and a six-way injection valve (1.5 mu L sample loop), but also to supply a volume pool of anode buffer so that the process of injection, focusing and mobilization of samples could be sequentially performed. The short-off valve in the tee unit or cross-shaped unit could be used to control the direction of the fluid flow. Using this online cIEF platform and under the optimized conditions, 7-proteins mixture could be separated and a good linear correlation between pI values and migration times was obtained by the M-IPG column. Meanwhile, based on the online cIEF platform, human serum proteins and a mixture of Hb A and Hb A(1c) have been successfully resolved with the newly developed M-IPG column.