title: Exonuclease-mediated ELISA-like assay for detecting DNA-binding activity of transcription factors: measurement of activated NF-kappaB.
abstract: This paper describes an exonuclease-mediated enzyme-linked immunosorbent assay (ELISA)-like assay (EMEA) for detecting the DNA binding activity of nuclear factor kappaB (NF-kappaB). For EMEA, a special double-stranded DNA (dsDNA)-coupled plate was first prepared by immobilizing a DNA probe on an N-oxysuccinimide ester-coated plate. The immobilized DNA probe, which was internally labeled with digoxigenin (DIG)-dT contained a NF-kappaB binding consensus sequence for capturing activated NF- kappaB in analyzed samples. For measurement, the plate was first incubated with a protein sample and then treated with exonuclease III to eliminate the probes not bound by NF-kappaB. Finally, the probes protected by NF-kappaB were colorimetrically detected by an alkaline phosphatase (AP)-conjugated anti-DIG antibody. The major advantage of EMEA is that it detects NF-kappaB without the need for NF-kappaB antibodies. EMEA may provide a general approach for assays of DNA sequence-specific transcription factors for which specific antibodies are unavailable, expensive, or of insufficient quality.
authors.affiliation: State Key laboratory of Bioelectronics, Southeast University, Nanjing, China. wangjinke@seu.edu.cn
authors.forename: Jinke
authors.initials: J
authors.lastname: Wang
authors.forename: Min L
authors.initials: ML
authors.lastname: Li
authors.forename: Dong
authors.initials: D
authors.lastname: Hua
authors.forename: Qixin
authors.initials: Q
authors.lastname: Chen
mesh_terms: D001483:Base Sequence; D001681:Biological Assay / Q000379:methods; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005092:Exonucleases; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D009841:Oligonucleotides; D014157:Transcription Factors; D004247:DNA; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C020133:exodeoxyribonuclease III