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Assay of DNA-binding proteins with a dsDNA-coupled plate
- Source :
- Clinical biochemistry. 39(2)
- Publication Year :
- 2005
-
Abstract
- Objectives: 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). Design and 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-κB, AP1 and SP1, and HeLa cell nuclear extract and assays of DNA-binding activity of NF-κB 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.
- Subjects :
- Chromatography
biology
Oligonucleotide
Chemistry
Sp1 Transcription Factor
Clinical Biochemistry
NF-kappa B p50 Subunit
General Medicine
Plasma protein binding
DNA
biology.organism_classification
DNA-binding protein
Primary and secondary antibodies
HeLa
Transcription Factor AP-1
chemistry.chemical_compound
Biochemistry
Covalent bond
biology.protein
Bioassay
Humans
Biological Assay
HeLa Cells
Protein Binding
Subjects
Details
- ISSN :
- 00099120
- Volume :
- 39
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Clinical biochemistry
- Accession number :
- edsair.doi.dedup.....5329d855b5d338635152d2feb379c41c