An enzyme-linked immunosorbent assay for the detection of agents which interfere with the DNA binding activities of transcription factors--exemplified by NF-IL6.
Binding of transcription factors to DNA is usually detected by the electrophoretic mobility shift assay also known as gel-shift or band-shift assay. Recently the use of biosensors has allowed factor/DNA interactions to be followed in real time. However, neither of these approaches lends itself easily to high-throughput screening of agents which might interfere with this process. We have therefore developed a 96-well plate-based enzyme-linked immunosorbent assay for this purpose. Biotinylated oligonucleotides bound to streptavidin-coated plates are used to capture recombinant transcription factor proteins which can then be detected using specific antibodies which in turn are recognised by peroxidase-conjugated antisera. The peroxidase catalyzes conversion of a colorless substrate to a colored product which can be quantified by optical densitometry on a plate reader. Agents which interfere with the binding of the transcription factor to DNA reduce the optical density in the well. Using the NF-IL6 transcription factor we show that the assay can detect reductions to 86% at significant levels. The assay may be readily adapted for robotic manipulation, making it ideal for high-throughput screening.
McKay
I A
IA
Centre for Cutaneous Research, London, England. i.a.mckay@mds.qmw.ac.uk
Kirby
L
L
Volyanik
E V
EV
Kumar
V
V
Wong
P W
PW
Bustin
S A
SA
D001483:Base Sequence; D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D051743:CCAAT-Enhancer-Binding Protein-delta; D004247:DNA