Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay.
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.
Fried
M G
MG
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
D001667:Binding, Competitive; D002021:Buffers; D004247:DNA / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D013237:Stereoisomerism; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism*
D002021:Buffers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA