Variables affecting the selectivity and efficiency of retention of DNA fragments by E. coli RNA polymerase in the nitrocellulose-filter-binding assay.
In this paper we characterize the effect of varying the solution conditions and filter-binding protocols on the extent and selectivity of DNA retention on nitrocellulose filters by DNA-binding proteins. These effects are illustrated by the binding interaction of Escherichia coli RNA polymerase with lambda and T7 phage DNA restriction fragments. We present procedures which will help enhance the selective retention of some DNA restriction fragments over others. These include increasing the pH and salt concentration, decreasing the enzyme-to-DNA ratio, and including an appropriate washing step. Selective binding is not dependent on the presence of Mg2+. Although we only show data for RNA polymerase-DNA interactions, many of the principles discussed are likely to find practical applications in studying selective DNA-protein binding in general.
D002352:Carrier Proteins / Q000378:metabolism; D003101:Collodion; D004247:DNA / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli; D005374:Filtration / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009876:Operon; D009994:Osmolar Concentration; D011485:Protein Binding; D012492:Salts
D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012492:Salts; D003101:Collodion; D004247:DNA; D012321:DNA-Directed RNA Polymerases