Assay of DNA-binding proteins with a dsDNA-coupled plate

Objectives:This paper fabricated a cost-effective dsDNA-coupled plate (dcPlate) and applied it to measure the abundance and DNA-binding activity of a DNA-binding protein (DBP).Design and Methods:The dcPlate was manufactured by covalently immobilizing an amino-modified oligonucleotide in wells of the plate coated with N-oxysuccinimide esters. The dcPlate was applied to measure the abundance of DNA-binding activity of a DBP in the same four steps, including protein incubation, primary antibody binding, enzyme-linked secondary antibody binding, and colorimetric development.Results:The detections of three purified DBPs including NF-κB, AP1 and SP1, and HeLa cell nuclear extract and assays of DNA-binding activity of NF-κB p50 to five various DNA sequences demonstrated that dcPlate can be used to measure the abundance of DBPs quantitatively and assay DNA-binding activity of DBPs in high throughputs format.Conclusions:The homemade cost-effective dcPlate provides a simple and versatile platform for studying DBPs.