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


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.

25 Reads
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The arthropod epidermis is an epithelium that deposits the apical cuticle, which is a stratified extracellular matrix (ECM) protecting the animal against pathogens, preventing dehydration and also serving as an exoskeleton. Differentiation of the cuticle conceivably implies coordinated production, secretion and localization of its components. The underlying molecular mechanisms are poorly explored. In this work, we show that the transcription factor Grainy head and the steroid hormone ecdysone drive the production of two partially overlapping sets of cuticle factors. Nevertheless, Grainy head is needed to modulate the expression of ecdysone signalling factors; the significance of this cross-talk is yet unclear. In addition, we found that ecdysone signalling negatively regulates its own impact. In conclusion, our findings suggest that at least two independently triggered pathways have evolved in parallel to cooperatively ensure the stereotypic implementation of the cuticle. As Grainy head is also essential for epithelial differentiation in vertebrates, we speculate that it acts to decode the ancient skin programme common to all animals. Full differentiation of the skin necessitates a second, complementing taxon-specific programme that requires its own decoder, which is represented by ecdysone in arthropods, whereas the vertebrate specific one remains to be identified.
    Insect Molecular Biology 03/2012; 21(3):283-95. DOI:10.1111/j.1365-2583.2012.01134.x · 2.59 Impact Factor

  • [Show abstract] [Hide abstract]
    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
Show more

Similar Publications