Efficient electroblotting of gel-resolved proteins onto diamond-like carbon-coated plate for protein-chip

Yoko Ino, Akiko Okayama, Y. Iwafune, Noriaki Arakawa, J. Kikuchi, M. Kamita, H. Kawasaki, Takeshi Okada, H. Hirano
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Abstract

We have developed a technique, which can produce high-density protein chips. In this technique, proteins are separated by gel electrophoresis, and electroblotted by semidry blotting apparatus onto a diamond-like carbon-coated stainless-steel plate (DLC plate) of which surface is modified chemically with amino groups. Proteins immobilized on this protein chip can interact with other proteins in solution, and proteins interacted with the immobilized proteins can be identified by mass spectrometric analysis. However, the electroblotting efficiency was not stable. We anticipated that the unstable efficiency might be related to the fluctuation of temperature during electroblotting. In the present study, to investigate the optimal temperature for efficient and effective electroblotting, we developed a semidry blotting apparatus, which could regulate blotting temperature from 4 to 45°C (±1°C). The high and reproducible blotting efficiency was obtained at not low temperature but rather high temperature (30°C). The temperature did not impede protein identification or analysis of protein-protein interactions on the DLC plates by mass spectrometry.
高效电印迹凝胶溶解蛋白质到类金刚石碳涂层板的蛋白质芯片
我们已经开发出一种技术,可以生产高密度的蛋白质芯片。在这项技术中,蛋白质通过凝胶电泳分离,并通过半干印迹装置在表面用氨基进行化学修饰的类金刚石碳涂层不锈钢板(DLC板)上进行电印迹。固定在该蛋白质芯片上的蛋白质可以与溶液中的其他蛋白质相互作用,并且与固定蛋白质相互作用的蛋白质可以通过质谱分析进行鉴定。然而,电印迹的效率并不稳定。我们推测这种不稳定的效率可能与电印迹过程中温度的波动有关。在本研究中,为了研究高效和有效的电印迹的最佳温度,我们开发了一种半干印迹装置,可以调节印迹温度从4到45°C(±1°C)。在高温(30°C)而非低温条件下获得了较高的重现性印迹效率。温度不影响蛋白质鉴定或质谱分析DLC板上蛋白质-蛋白质相互作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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