Rapid qualitative evaluation of DNA transcription factor NF-κB by microchip electrophoretic mobility shift assay in mammalian cells

Sonoko Inoue, Noritada Kaji, Masatoshi Kataoka, Yasuo Shinohara, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba

研究成果: Contribution to journalArticle査読

7 被引用数 (Scopus)

抄録

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 (~200s) 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 5min. Furthermore, without any purification process, even crude NF-κB in nuclear extracts from HeLa cells was specifically detected within 120s by the μEMSA.

本文言語英語
ページ(範囲)3241-3247
ページ数7
ジャーナルELECTROPHORESIS
32
22
DOI
出版ステータス出版済み - 11 2011
外部発表はい

All Science Journal Classification (ASJC) codes

  • 分析化学
  • 生化学
  • 臨床生化学

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