title: Rapid qualitative evaluation of DNA transcription factor NF-κB by microchip electrophoretic mobility shift assay in mammalian cells.
abstract: 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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima, Japan.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Inoue
authors.forename: Noritada
authors.initials: N
authors.lastname: Kaji
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Kataoka
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Manabu
authors.initials: M
authors.lastname: Tokeshi
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Baba
mesh_terms: D002232:Carbocyanines; D002457:Cell Extracts / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D046209:Electrophoresis, Microchip / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000737:chemistry* / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
chemical_list: D002232:Carbocyanines; D002457:Cell Extracts; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; C085321:cyanine dye 5