Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection.
Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection.
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DOI：
10.1002/elps.201000151
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发表时间：
2010-08
期刊：
影响因子：
2.9
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
--
作者：
Dickerson, Jane A.;Ramsay, Lauren M.;Dada, Oluwatosin O.;Cermak, Nathan;Dovichi, Norman J.
毛细管等电聚焦和毛细管区带电泳与激光诱导荧光检测相结合，建立了一种超灵敏的蛋白质二维分离方法。在该方法中，两个毛细管通过缓冲液填充的界面连接。单独的电源控制第一毛细管进样端和接口处的电位;检测器保持在地电位。蛋白质用荧光试剂Chromeo P503标记，其保留标记蛋白质的等电点。将标记的蛋白质与两性电解质混合并注入第一维毛细管中。在高pH下毛细管的注射端和低pH下的界面进行聚焦步骤。为了使组分流动，界面填充有与第二维分离相容的高pH缓冲液。将级分转移到第二维毛细管中进行分离。馏分转移和第二维分离的过程重复了二十多次。分离产生了125个点的容量。
Capillary isoelectric focusing and capillary zone electrophoresis are coupled with laser-induced fluorescence detection to create an ultrasensitive two-dimensional separation method for proteins. In this method, two capillaries are joined through a buffer filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second dimension separation. A fraction was transferred to the second dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125.
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