Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfludic network
Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfludic network
复制标题
DOI：
10.1021/ac034765b
复制
发表时间：
2004-02-01
影响因子：
7.4
通讯作者：
Lee, CS
中科院分区：
文献类型：
--
作者：
Li, Y;Buch, JS;Lee, CS
据报道，一种集成的蛋白质浓缩/分离系统，在聚合物微流控芯片上将非天然等电聚焦（IEF）与十二烷基硫酸钠（SDS）凝胶电泳相结合。该系统为分离的蛋白质混合物提供显着的分析物浓度和极高的分辨率。在塑料微流体网络中引入和分离多种分离介质的能力是实现多维蛋白质分离的两个关键要求之一。第二个要求在于聚焦蛋白质从第一分离维度到第二分离维度的定量转移，而从第一维度获得的分辨率没有显着损失。不是按顺序对从 IEF 洗脱的蛋白质分析物进行取样，而是将聚焦的蛋白质通过电动转移到一系列正交微通道中，并通过 SDS 凝胶电泳以平行和高通量的形式进一步解析。使用非共价、环境敏感的荧光探针（例如 Sypro Red）监测解析的蛋白质分析物。与共价标记蛋白质相比，在电泳分离过程中使用 Sypro 染色不仅为分析细胞裂解物等复杂蛋白质混合物提供了一种通用检测方法，而且还避免了标记反应导致的蛋白质微异质性的额外引入。使用平面尺寸小至 2 cm x 3 cm 的芯片，可在 10 分钟内完成全面的 2-D 蛋白质分离，总峰容量接近 1700。通过简单地提高阵列中微通道的密度，可以实现峰容量的显着增强，从而增加在基于尺寸的分离维度中进一步分析的 IEF 级分的数量。
An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of similar to1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.