DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.
AUTOR(ES)
Xian, J
RESUMO
A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=40183Documentos Relacionados
- High-sensitivity troponins: a major breakthrough
- Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis
- The DNA-protein cross: a method for detecting specific DNA-protein complexes in crude mixtures.
- High-Sensitivity C-Reactive Protein and Cardiovascular Disease Across Countries and Ethnicities
- Minichromosomal repetitive DNA in Trypanosoma cruzi: its use in a high-sensitivity parasite detection assay.