Detection of cyclic di-GMP binding proteins utilizing a biotinylated cyclic di-GMP pull-down assay

Cyclic di-GMP is an important regulatory messenger molecule that often directly interacts with proteins to alter function. It is therefore important to find potential c-di-GMP binding proteins and verify a direct interaction between them. Here, we describe a pull-down assay using biotinylated-c-di-GMP to capture a specific protein of interest followed by immunoblot analysis to determine relative protein abundance. This method also allows for addition of both specific and nonspecific competitors to determine specificity of c-di-GMP–protein binding. We also discuss using densitometry analysis on resulting immunoblots to calculate the dissociation constant (K_D) of the binding reaction, allowing for a determination of binding affinity.