Fully automated two-dimensional electrophoresis system for high-throughput protein analysis
Fully automated two-dimensional electrophoresis system for high-throughput protein analysis
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DOI：
10.1021/ac070485a
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发表时间：
2007-08-01
影响因子：
7.4
通讯作者：
Yokoyama, Kenji
中科院分区：
文献类型：
--
作者：
Hiratsuka, Atsunori;Kinoshita, Hideki;Yokoyama, Kenji
我们开发了一种全自动电泳系统，用于快速和高重复性的蛋白质分析。等电聚焦（IEF）、局部蛋白染色、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、原位蛋白检测等二维（2D）电泳均自动完成。该系统由Peltiert装置、高压发电装置、电极和三个一次性聚甲基丙烯酸甲酯（PMMA）部件组成，用于IEF、反应室和SDS-PAGE。由于IEF部件的小型化，可以在30分钟内实现快速IEF。在SDS-PAGE部件上使用锥形边缘凝胶实现了部件之间的连接，而无需使用粘合材料。采用双轴输送机进行零件定位、样品导入和洗涤过程，实现了低维护和低成本的自动化系统。在检测数量、分辨率和蛋白质斑点的再现性方面，比较了该系统和商用minigel系统的性能。尽管聚焦时间更短，零件尺寸更小，但该系统实现了与minigel系统相当的高分辨率。结果再现性优于或可与minigel系统的性能相媲美。在1.5小时内完成二维分离。该系统实用、便携，并具有自动化功能。
We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. 'Me resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities.