title: Design and optimization of a capillary electrophoretic mobility shift assay involving trp repressor-DNA complexes.
abstract: An investigation of DNA-protein interactions by capillary electrophoresis (CE) with laser fluorometric detection is performed that combines the rapid and minimal sample consumption methods of CE with the selective separation influence of mobility shift assays. An inspection of the well characterized interaction between the trp repressor of Escherichia coli and the trp operator (DNA) is the basis of the assay. The use of fluorescently tagged operator not only lends itself to laser-induced fluorescence detection but also precludes the use of radiolabeled detection. It is demonstrated that composition and pH of the running buffer are critical for maximized efficiency and resolution of operator from the repressor-operator complex. Quantitative studies showed reaction of repressor with operator resulted in the diminishing of free operator signal and the simultaneous creation of the repressor-operator peak that is well resolved from the free operator. Also examined was the ability to perform qualitative studies involving non-specific interactions between the operator and a complex protein sample. It is shown that the specificity of operator for repressor can be used to selectively separate the repressor from a complex sample that includes non-specific proteins.
authors.affiliation: Department of Chemistry, University of Tennessee, Knoxville 37996-1600, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Stebbins
authors.forename: A M
authors.initials: AM
authors.lastname: Hoyt
authors.forename: M J
authors.initials: MJ
authors.lastname: Sepaniak
authors.forename: B K
authors.initials: BK
authors.lastname: Hurlburt
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D029968:Escherichia coli Proteins
chemical_list: D001426:Bacterial Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins