Article

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

State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
Clinical Biochemistry (Impact Factor: 2.28). 03/2006; 39(2):167-75. DOI: 10.1016/j.clinbiochem.2005.10.017
Source: PubMed

ABSTRACT 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).
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.
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.
The homemade cost-effective dcPlate provides a simple and versatile platform for studying DBPs.

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