Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima, Japan.
Electrophoresis. 2011 Nov;32(22):3241-7. doi: 10.1002/elps.201100261.
We have developed a separation technique for DNA-protein complex based on electrophoretic mobility shift assay (EMSA) by microchip electrophoresis, which we call microchip electrophoretic mobility shift assay (μEMSA). To evaluate the μEMSA, we employed recombinant human nuclear factor-κB (rhNF-κB) and its consensus double-stranded oligonucleotide (dsOligo) fluorescently labeled with Cy5. We carried out the electrophoretic separation of the consensus dsOligo-rhNF-κB complex and the unbound dsOligo in methylcellulose solution and confirmed rapid (∼200 s) and reliable identification and semi-quantitation of the specific interaction between dsOligo and rhNF-κB. The binding specificity of rhNF-κB was confirmed by introducing non-fluorescently labeled consensus oligonucleotide as a competitor. The progression of the binding reaction under various incubation times was monitored, and it was found that the dsOligo and rhNF-κB complex formation reached equilibrium (ca. 90% of the dsOligo was bound to rhNF-κB) after 5 min. Furthermore, without any purification process, even crude NF-κB in nuclear extracts from HeLa cells was specifically detected within 120 s by the μEMSA.
我们开发了一种基于电泳迁移率变动分析（EMSA）的微芯片电泳分离技术，我们称之为微芯片电泳迁移率变动分析（μEMSA）。为了评估μEMSA，我们使用了重组人核因子-κB（rhNF-κB）及其与 Cy5 荧光标记的双链寡核苷酸（dsOligo）。我们在甲基纤维素溶液中进行了共识 dsOligo-rhNF-κB 复合物与未结合的 dsOligo 的电泳分离，并证实了快速（约 200 s）和可靠地识别和半定量 dsOligo 与 rhNF-κB 之间的特异性相互作用。通过引入非荧光标记的共识寡核苷酸作为竞争物，证实了 rhNF-κB 的结合特异性。监测了不同孵育时间下结合反应的进展，发现 dsOligo 和 rhNF-κB 复合物形成在 5 分钟后达到平衡（约 90%的 dsOligo 与 rhNF-κB 结合）。此外，无需任何纯化过程，甚至可以在 120 s 内通过 μEMSA 特异性检测来自 HeLa 细胞核提取物中的粗制 NF-κB。
