Assay of DNA-binding proteins with a dsDNA-coupled plate
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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ABSTRACT: This study describes a simple method for analyzing the abundance of mRNA molecules in a total DNA sample. Due to the dependence on the near-infrared fluorescence technique, this method is named near-infrared fluorescence gene expression detection (NIRF-GED). The procedure has three steps: (1) isolating total RNA from detected samples and reverse-transcription into cDNA with a biotin-labeled oligo dT; (2) hybridizing cDNA to oligonucleotide probes coupled to a 96-well microplate; and (3) detecting biotins with NIRF-labeled streptavidin. The method was evaluated by performing proof-in-concept detections of absolute and relative expressions of housekeeping and NF-κB target genes in HeLa cells. As a result, the absolute expression of three genes, Ccl20, Cxcl2, and Gapdh, in TNF-α-uninduced HeLa cells was determined with a standard curve constructed on the same microplate, and the relative expression of five genes, Ccl20, Cxcl2, Il-6, STAT5A, and Gapdh, in TNF-α-induced and -uninduced HeLa cells was measured by using NIRF-GED. The results were verified by quantitative PCR (qPCR) and DNA microarray detections. The biggest advantage of NIRF-GED over the current techniques lies in its independence of exponential or linear amplification of nucleic acids. Moreover, NIRF-GED also has several other benefits, including high sensitivity as low as several fmols, absolute quantification in the range of 9 to 147 fmols, low cDNA consumption similar to qPCR template, and the current medium throughput in 96-well microplate format and future high throughput in DNA microarray format. NIRF-GED thus provides a new tool for analyzing gene transcripts and other nucleic acid molecules.Analytical and Bioanalytical Chemistry 12/2013; 406(2). DOI:10.1007/s00216-013-7486-9 · 3.58 Impact Factor
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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 · 2.98 Impact Factor