ABSTRACT
Over the last three decades, the kinetics of many systems have been investigated at the single-molecule level. An important consideration for any assay is its specificity and the degree to which the results may be affected by contaminants. Assays designed to follow kinetics in vivo bypass the purification problem by using a specific probe to report on one or a limited number of reactions. Filter binding assays can be used to evaluate equilibrium constants. Equilibrium constants and stoichiometries may be determined in two ways: direct and indirect. In the first case, the concentrations of the components at equilibrium are measured in terms of molarity. In the second case, a physical parameter is measured that distinguishes free and bound states. Equilibrium binding constants can also be determined using competition assays in which the binding of labeled nucleic acid to a protein is inhibited by the presence of excess unlabeled nucleic acid.
