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
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.
Available from: umesh gangishetti
- "The assays for the affinity of the DNA-binding domains of Grh and bFtzF1 to oligomers harbouring predicted recognition sequences in the promoter region of putative target genes were based on the nonradioactive protocol developed by Wang et al. (2006). The DNA-binding domains of Grh (Uv et al., 1994) and bFtzF1 (Ohno et al., 1994) were fused to glutathione S-transferase (GST) in the expression vector pGex-4T1 (GE Healthcare, Fairfield, CT, USA). "
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Insect Molecular Biology 03/2012; 21(3):283-95. DOI:10.1111/j.1365-2583.2012.01134.x · 2.59 Impact Factor
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ABSTRACT: 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-kappaB. The optimized parameters of dcPlate for assay of NF-kappaB were as follows: immobilized DNA probe at the concentration of 25 pmol/100 microL-well, incubation time of 90 min for NF-kappaB binding to dcPlate, primary and secondary antibody concentration of 0.1 microL/100 microL dilution, incubation time of 90 min for primary antibody binding to NF-kappaB, temperature of 25 degrees 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-kappaB p50 and the cell nuclear extract were from 0.59 to 75 ng/well and 0.313 to 10 microg/well, respectively.
Colloids and surfaces B: Biointerfaces 04/2007; 55(1):31-7. DOI:10.1016/j.colsurfb.2006.11.015 · 4.15 Impact Factor
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