DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.
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.
Xian
J
J
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Harrington
M G
MG
Davidson
E H
EH
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010063:Ovum / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012617:Sea Urchins; D014157:Transcription Factors / Q000737:chemistry*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; C072372:P3A2 regulatory protein, Strongylocentrotus purpuratus