Miniaturized two-dimensional gel electrophoresis of high-molecular-weight proteins using low-concentration multifilament-supporting gel for isoelectric focusing

T. Odake, T. Saheki, K. Kamezawa, Kozue Miyata, H. Takiguchi, H. Hotta, Onda Koki, Yasuaki Kojima, Jinxiang Li, K. Tsunoda
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Abstract

A miniaturized two-dimensional gel electrophoresis (2-DE) system was constructed with a multifilament-supporting (MFS) gel as a first-dimensional gel for isoelectric focusing (IEF). The MFS gel was 1 mm in diameter, had a multifilament yarn of acrylic as a gel-support, and could provide low-concentration (2.5%T) polyacrylamide with sufficient mechanical strength, resulting in fast IEF, and clear 2-D spots due to the relatively large pore and improved transfer efficiency from the first-dimensional gel to the second-dimensional one of especially high-molecular-weight (HMW) proteins. In this study, by using the MFS gel of 4 cm in length as a miniaturized IEF gel, three colored proteins were separated in 8 min, which was about twice faster than using the MFS gel of standard length (7 cm). The second-dimensional gel was also miniaturized to be 55 mm wide, 40 mm long and 0.75 mm thick. The low-concentration MFS gel after IEF was easily transferred onto the top of the miniaturized second-dimensional gel with a tweezers. By using the miniaturized 2-DE system, the HMW size marker was separated in 30 min by IEF and in 25 min by native-polyacrylamide gel electrophoresis (Native-PAGE), which was at least twice faster than by using the standard size 2-DE system using MFS gel. This miniaturized 2-DE system could be expected as a useful separation method for fast protein diagnosis and screening.
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使用低浓度多丝支持凝胶进行等电聚焦的高分子量蛋白质的小型化二维凝胶电泳
采用多丝支撑(MFS)凝胶作为等电聚焦(IEF)的一维凝胶,构建了小型化的二维凝胶电泳(2-DE)系统。MFS凝胶直径为1mm,以丙烯酸多丝纱为凝胶支撑,可提供具有足够机械强度的低浓度(2.5%T)聚丙烯酰胺,具有快速的IEF,且由于孔隙较大,二维斑点清晰,提高了高分子量(HMW)蛋白质从一维凝胶到二维凝胶的传递效率。在本研究中,使用长度为4 cm的MFS凝胶作为微型IEF凝胶,在8 min内分离了3个有色蛋白,比使用标准长度(7 cm)的MFS凝胶快了约2倍。二维凝胶也被小型化为55毫米宽,40毫米长,0.75毫米厚。IEF后的低浓度MFS凝胶可以很容易地用镊子转移到微缩的二维凝胶的顶部。使用小型化的2-DE体系,用IEF和Native-PAGE分别在30 min和25 min内分离了HMW大小标记物,比使用MFS凝胶的标准尺寸2-DE体系分离速度快至少两倍。这种小型化的2-DE系统有望成为一种有效的蛋白质快速诊断和筛选分离方法。
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