An optimized assay for transcription factor NF-κB with dsDNA-coupled microplate

To develop an EMSA-free assay approach for analyzing the sequence-specific DNA-binding proteins (DBPs), an easy cost-effective dsDNA-coupled plate (dcPlate) was developed in our lab for this purpose. In this paper, the assay conditions of such dcPlate were fully optimized for detecting an important transcription factor, NF-κB. The optimized parameters of dcPlate for assay of NF-κB were as follows: immobilized DNA probe at the concentration of 25 pmol/100 μL-well, incubation time of 90 min for NF-κB binding to dcPlate, primary and secondary antibody concentration of 0.1 μL/100 μL dilution, incubation time of 90 min for primary antibody binding to NF-κB, temperature of 25 °C for the above process, colorimetric developing time for 30 min. After optimization, the signal was improved three times higher than that from not optimized conditions. The linear colorimetric detection ranges of the purified recombinant NF-κB p50 and the cell nuclear extract were from 0.59 to 75 ng/well and 0.313 to 10 μg/well, respectively.