Fried M G
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Electrophoresis. 1989 May-Jun;10(5-6):366-76. doi: 10.1002/elps.1150100515.
Native gel electrophoresis (mobility shift) assays may be used to obtain quantitative information about the site distribution, equilibria and kinetics of protein-DNA interactions. These applications depend on the ability of the electrophoretic system to resolve the reaction components, and on their stabilities during the separation process. Factors which affect the lifetimes and mobilities of protein-DNA complexes during electrophoresis include reaction and electrophoresis buffer composition, pH, and ionic strength; the presence of low molecular weight effectors and enzymatic substrates; the nature and concentration of the gel matrix; the temperature; the molecular weights of protein and DNA; the stoichiometric ratios of their complexes; and the possibility of conformational and configurational isomerization of reaction components. We discuss how these factors influence the acquisition of quantitative data from electrophoretic patterns and band intensities, and present formulas for the estimation of equilibrium constants and rate constants for prototypical DNA-protein interactions.
天然凝胶电泳（迁移率变动）分析可用于获取有关蛋白质 - DNA 相互作用的位点分布、平衡和动力学的定量信息。这些应用取决于电泳系统分辨反应组分的能力，以及它们在分离过程中的稳定性。在电泳过程中影响蛋白质 - DNA 复合物寿命和迁移率的因素包括反应和电泳缓冲液的组成、pH 值和离子强度；低分子量效应物和酶底物的存在；凝胶基质的性质和浓度；温度；蛋白质和 DNA 的分子量；它们复合物的化学计量比；以及反应组分构象和构型异构化的可能性。我们讨论了这些因素如何影响从电泳图谱和条带强度获取定量数据，并给出了用于估计典型 DNA - 蛋白质相互作用的平衡常数和速率常数的公式。
