Assay of DNA-binding proteins with a dsDNA-coupled plate.
OBJECTIVE
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).


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-kappaB, AP1 and SP1, and HeLa cell nuclear extract and assays of DNA-binding activity of NF-kappaB 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.
Wang
Jin K
JK
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China. wangjinke@seu.edu.cn
Li
Jian L
JL
Li
Min L
ML
Hua
Dong
D
Chen
Hong M
HM
D001681:Biological Assay; D004247:DNA / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D052002:NF-kappa B p50 Subunit / Q000378:metabolism*; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D052002:NF-kappa B p50 Subunit; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D004247:DNA