Open AccessExonuclease-mediated ELISA-like assay for detecting DNA-binding activity of transcription factors: measurement of activated NF-κB
Author(s) -
Jinke Wang,
Min L. Li,
Honglin Dong,
Qixin Chen
Publication year - 2006
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000112218
Subject(s) - exonuclease , dna , digoxigenin , exonuclease iii , microbiology and biotechnology , alkaline phosphatase , hybridization probe , biology , transcription (linguistics) , transcription factor , antibody , chemistry , enzyme , biochemistry , gene expression , dna polymerase , gene , in situ hybridization , genetics , escherichia coli , linguistics , philosophy
This paper describes an exonuclease-mediated enzyme-linked immunosorbent assay (ELISA)-like assay (EMEA) for detecting the DNA binding activity of nuclear factor κB (NF-κB). 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-κB binding consensus sequence for capturing activated NF-κB 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-κB. Finally, the probes protected by NF-κB were colorimetrically detected by an alkaline phosphatase (AP)-conjugated anti-DIG antibody. The major advantage of EMEA is that it detects NF-κB without the need for NF-κB 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.