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  • 1
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 1535-1536 
    ISSN: 0173-0835
    Keywords: Mobility-shift assay ; Nonspecific protein-DNA complexes ; Gel cage ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Theoretical mobility-shift patterns are computed by solution of conservation equations for electrophoresis coupled with chemical reaction. The chemical reaction term is often formulated in terms of dissociation of the protein-DNA complex in a gel cage. This formulation assumes that once the dissociated protein escapes the cage, it goes down a sink and is totally lost. This implies that the concentration of the escaped protein is too low to affect sign ficantly the rates of protein-DNA association along its migration pathway.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
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  • 2
    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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  • 3
    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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