Fully Automated Two-Dimensional Electrophoresis System for High-Throughput Protein Analysis

• Published in: Analytical chemistry (Washington) 2007-08, Vol.79 (15), p.5730-5739
• Main Authors: Hiratsuka, Atsunori, Kinoshita, Hideki, Maruo, Yuji, Takahashi, Katsuyoshi, Akutsu, Satonari, Hayashida, Chie, Sakairi, Koji, Usui, Keisuke, Shiseki, Kisho, Inamochi, Hajime, Nakada, Yoshiko, Yodoya, Kouhei, Namatame, Ichiji, Unuma, Yutaka, Nakamura, Makoto, Ueyama, Kosuke, Ishii, Yoshinori, Yano, Kazuyoshi, Yokoyama, Kenji
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Automation; Chemistry; Chromatographic methods and physical methods associated with chromatography; Electric fields; Electrophoresis, Gel, Two-Dimensional - methods; Exact sciences and technology; Isoelectric Focusing - methods; Molecules; Proteins; Proteins - analysis; Proteins - chemistry; Reproducibility of Results; Sodium Dodecyl Sulfate - chemistry; Staining and Labeling
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac070485a

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. The 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.