Studies of protein--DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization.
Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 x 10(6) M(-1)) and the 37-mer (23 x 10(6) M(-1)). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein-DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.
Wan
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Environmental Health Sciences Program, Department of Public Health Sciences, University of Alberta, Edmonton, Canada.
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D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D007834:Lasers; D011485:Protein Binding
D017931:DNA Primers; D004268:DNA-Binding Proteins; D004247:DNA