Abstract
Modulation of signal transduction via binding of the secondary messenger molecule cyclic di-GMP to effector proteins is a near universal regulatory schema in bacteria. In particular, direct binding of c-di-GMP to transcriptional regulators has been shown to alter gene expression of a variety of processes. Here, we illustrate a pull-down-based DNA:protein binding reaction to determine the relative importance of c-di-GMP in the binding affinity of a target protein to specific DNA sequences. Specifically, the pull-down-based assay enables DNA binding to be analyzed with differing concentrations of c-di-GMP in the absence/presence of specific and nonspecific competitors.