Li Qian, Zhao Xinying, Liu Hongyang, Qu Feng
School of Life Science, Beijing Institute of Technology, Beijing 100081, China.
Beijing Centre for Physical and Chemical Analysis, Beijing 100089, China.
J Chromatogr A. 2014 Oct 17;1364:289-94. doi: 10.1016/j.chroma.2014.08.073. Epub 2014 Aug 27.
In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) as a CE-SELEX variant is proposed. Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein are incubated with a FAM labeled ssDNA library, respectively. Incubation mixture is separated in low pH CE (pH 2.6), where positively charged protein, protein-ssDNA complex and negatively charged ssDNA library migrate oppositely without EOF driven. Analysis of protein-ssDNA complex under positive voltage and unbound ssDNA library under negative voltage by CE-UV are applied for interactive evaluation. By increasing injection time, larger amount protein-ssDNA complex can be collected conveniently at the cathode end whereas ssDNA migrates to anode. Finally, stability of protein-ssDNA complex in low pH CE separation is discussed.
在这项工作中,提出了一种新型的低pH毛细管电泳-指数富集配体系统(LpH-CE-SELEX)作为毛细管电泳-指数富集配体系统的变体。分别将转铁蛋白(Trf)、牛血清白蛋白(BSA)和细胞色素c(Cyt c)作为模型蛋白与荧光素标记的单链DNA文库孵育。孵育混合物在低pH毛细管电泳(pH 2.6)中进行分离,其中带正电荷的蛋白质、蛋白质-单链DNA复合物和带负电荷的单链DNA文库在没有电渗流驱动的情况下向相反方向迁移。通过毛细管电泳-紫外检测,在正电压下分析蛋白质-单链DNA复合物,在负电压下分析未结合的单链DNA文库,用于相互作用评估。通过增加进样时间,可以在阴极端方便地收集到大量的蛋白质-单链DNA复合物,而单链DNA则迁移到阳极。最后,讨论了蛋白质-单链DNA复合物在低pH毛细管电泳分离中的稳定性。