Theoretical studies on the mobility‐shift assay of protein‐DNA complexes

The theory of mass transport coupled to reversible macromolecular interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility-shift behavior of protein-DNA complexes. Model systems include (i) specific binding of a univalent protein molecule to a single site on the DNA molecule; (ii) the putative cage effect; (iii) cooperative binding to multiple sites; (iv) formation of looped complexes of 1:1 and 2:1 stoichiometry; (v) noncooperative and cooperative, nonspecific binding modes; and (vi) binding of dimerizing transcriptional factors to response elements of target genes. Favorable comparison of simulated with experimental mobility-shift behavior indicates that the phenomenological mechanisms, whereby observed mobility-shift patterns are generated during electrophoresis, are embodied in the theory. These studies have provided guidelines for definitive interpretation of mobility-shift assays and for the design of experiments to develop a detailed understanding of the particular system under investigation.