A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) for isoelectric focusing of amino acids and proteins
A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) for isoelectric focusing of amino acids and proteins
复制标题
用于氨基酸和蛋白质等电聚焦的具有固定 pH 梯度的多孔层开管毛细管柱 (PLOT-IPG)
DOI：
10.1016/j.aca.2018.10.036
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发表时间：
2019-02-07
影响因子：
6.2
通讯作者：
Yan, Chao
中科院分区：
文献类型：
--
作者：
Liu, Rangdong;Cheddah, Soumia;Yan, Chao
采用两步制备了固定化pH梯度多孔层开管毛细管柱（PLOT IPG）。首先，通过丙烯酰胺、甲基丙烯酸缩水甘油酯和N，N ′-亚甲基双丙烯酰胺的原位聚合，合成了含有活性环氧基团的部分填充多孔层开管毛细管柱。然后，将商业载体两性电解质（CA，Pharmalyte）的水溶液聚焦在制备的聚合物柱中。用Pharmalyte 4.5-6.0（窄pH范围）或Pharmalyte 3.0-10.0（宽pH范围）固定的色谱柱，可分离已知pI的蛋白质，且pI值与迁移时间之间具有良好的线性相关性。同时，与传统梯度相比，分辨率得到了提高，且pH范围较宽。低分子量氨基酸的混合物与窄pH范围的PLOT-IPG柱分离;从所获得的结果，我们的新柱的分辨率计算为至多0.09 pH单位。此外，采用新开发的宽、窄pH范围PLOT IPG柱对人血清蛋白进行分析，其在窄pH范围内的灵敏度和分离度均优于毛细管电泳。(C)2018爱思唯尔B. V.保留所有权利。
A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) was prepared in two steps. First, a partially filled porous layer open tubular capillary column containing active epoxy groups was synthesized by in situ polymerization of acrylamide, glycidyl methacrylate, and N,N'-methylenebisacrylamide. Then, an aqueous solution of commercial carrier ampholytes (CAs, Pharmalyte) was focused in the prepared polymer column. With the column immobilized by Pharmalyte 4.5-6.0 (narrow pH range) or Pharmalyte 3.0-10.0 (wide pH range), proteins with known pIs were separated and a good linear correlation between pI values and migration times was obtained. Meanwhile, resolution was improved compared to the traditional gradient with a wide pH range. A mixture of low molecular weight amino acids was separated with the narrow pH range PLOT-IPG column; from the obtained results, the resolution of our new column was calculated to be at most 0.09 pH unit. In addition, human serum proteins were analyzed with the newly developed wide and narrow pH range PLOT-IPG columns; its sensitivity and resolution in the narrow pH range one were better than capillary electrophoresis. (C) 2018 Elsevier B.V. All rights reserved.