National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 116023 Dalian, PR China.
Electrophoresis. 2009 Dec;30(23):4034-9. doi: 10.1002/elps.200900209.
Monolithic immobilized pH gradient (M-IPG) materials were prepared in microchannles by photoinitiated polymerization of acrylamide, glycidylmethacrylate and Bis, followed by the attachment of focused Ampholine onto the surface of porous monoliths via epoxide groups. With M-IPG materials as matrix, FITC-labeled ribonuclease B, myoglobin and alpha-casein were well separated by microchip isoelectric focusing (muCIEF) without carrier amphocytes (CAs) added in the buffer. Both chemical and pressure mobilization were applied to drive focused zones for LIF detection. Our experimental results showed that pressure mobilization was preferable with neglectable band broadening, and good peak shape and high detection sensitivity were obtained. All these results demonstrate that muCIEF with M-IPG materials is not only an efficient mode for protein enrichment and separation but also attractive to couple with other CE modes to achieve multi-dimensional separation or MS for further identification, without the interference of mobile CAs.
整体式固定化 pH 梯度（M-IPG）材料通过丙烯酰胺、甲基丙烯酰氧乙基二甲基氯化铵和双丙烯酰胺的光引发聚合在微通道中制备，然后通过环氧基将聚焦的 Ampholine 附着到多孔整体的表面上。以 M-IPG 材料为基质，在缓冲液中不加载体两性电解质（CA）的情况下，通过微芯片等电聚焦（μCIEF）可以很好地分离 FITC 标记的核糖核酸酶 B、肌红蛋白和α-酪蛋白。化学和压力迁移都被应用于驱动聚焦区进行 LIF 检测。我们的实验结果表明，压力迁移是优选的，几乎没有带宽展宽，并且可以获得良好的峰形和高检测灵敏度。所有这些结果表明，M-IPG 材料的 μCIEF 不仅是一种有效的蛋白质浓缩和分离模式，而且还可以与其他 CE 模式结合，实现多维分离或 MS 进行进一步鉴定，而不会受到迁移 CA 的干扰。
