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  • 1
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 127-141 
    ISSN: 0173-0835
    Keywords: Mobility-shift assay ; Retardation analysis ; Protein-DNA complexes ; Computer simulations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The theory of mass transport coupled to revesible macromolecular interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility-shift behavior of protein-DNA complexes. Model systems include (i) specific binding of a univalent protein molecule to a single site on the DNA molecule; (ii) the putative cage effect; (iii) cooperative binding to multiple sites; (iv) formation of looped complexes of 1:1 and 2:1 stoichiometry; (v) noncooperative and cooperative, nonspecific binding modes; and (vi) binding of dimerizing transcriptional factors to response elements of target genes. Favorable comparison of simulated with experimental mobility-shift behavior indicates that the phenomenological mechanisms, whereby observed mobility-shift patterns are generated during electrophoresis, are embodies in the theory. These studies have provided guidelines for definitive interpretation of mobility-shift assays and for the design of experiments to develop a detailed understanding of the particular system under investigation.
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1092-1097 
    ISSN: 0173-0835
    Keywords: Mobility-shift assay ; Retardation analysis ; Dimerizing protein-DNA complexes ; Computer simulations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The theory of mass transport coupled to macromolecular interactions under chemical kinetic control forms the basis of four different models of the electrophoretic mobility-shift assay of complexes formed between dimerizing proteins and DNA. The theory of mass action was applied to the set of simultaneous dimerization (either simple or ligand-induced) and DNA-binding reactions in order to fix the initial equilibrium composition of mixtures to be assayed. Theoretical mobility-shift patterns were obtained for a range of protein concentrations at constant DNA concentration by numerical solution of the set of simultaneous transport-reaction equations appropriate for each model. In those cases in which dimerization in solution is modeled (including heterodimerization), analysis of the peaks in the patterns provides apparent binding constants, which, when extrapolated to infinite dilution of protein, yield acceptable estimates of equilibrium constants. Those for binding of dimer are products of two or three equilibrium constants, from which the equilibrium binding constant can be extracted, privided that dimerization and, where required, ligand-binding constants are determined by independent physicochemical methods. Dimerization of protein when bound to DNA is distinctive in that extrapolation to infinite dilution of protein is not required.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0173-0835
    Keywords: Mobility-shift assay ; Retardation analysis ; Nonspecific protein-DNA complexes ; Conditional probabilities ; Gel cage ; Histone-like bacterial protein (HU) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Complexes of an 88 bp DNA and the HU protein were studied by both experimental and theoretical electrophoretic mobility-shift analyses. Experimental analysis defined the stoichiometry of binding and estimated an apparent intrinsic dissociation constant (Kd = 1 to 3 × 10-7 M) for the HU:DNA complexes. The theory of conditional probabilities was applied to the binding of HU to DNA in order to fix the initial equilibrium composition of mixtures to be assayed theoretically by the mobility-shift procedure. Electrophoretic mobility-shift patterns were obtained by numerical solution of a set of simultaneous transport-reaction equations, in which the chemical kinetic term is formulated in terms of dissociation of the different DNA:HU complexes in gel cages. The computed patterns simulated the experimental patterns describing the titration of a fixed concentration of an 88 bp DNA fragment with dimeric HU. These insightful results provide guidelines for interpretation of the electrophoretic behavior of systems in which a ligand binds nonspecifically to DNA. In particular, the narrow unresolved zone observed both experimentally and theoretically beyond 50-60% saturation is a reaction zone characteristic of noncooperative ligand-binding governed by conditional probabilities. The discrepancy between the theoretically assigned and experimental values of the intrinsic binding constant is attributed to an HU-induced change in the conformation of DNA.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 12-19 
    ISSN: 0173-0835
    Keywords: Mobility-shift assay ; Retardation analysis ; Nonspecific protein-DNA complexes ; Histone-like bacterial protein ; Cooperative binding ; Gel cage ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The simulated electrophoretic mobility-shift behavior of a model system, in which the nonspecific binding of a protein to a DNA fragment is cooperative, was compared with the experimental behavior of the DNA: histone-like bacterial protein (HU) system. It was concluded that the binding of HU to an 88 bp DNA fragment is, at least, not highly cooperative. The theory of mobility-shift analysis was extended even further to encompass high affinity sequence-specific binding of protein to a DNA fragment followed by weak nonspecific binding, the latter governed by conditional probabilities. In addition to featuring a ladder of incremental protein-DNA complexes, the computed mobility-shift patterns placed emphasis upon stabilization of weak, nonspecific complexes in gel cages.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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