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 (KD) of the binding reaction, allowing for a determination of binding affinity.
CITATION STYLE
Chambers, J. R., & Sauer, K. (2017). Detection of cyclic di-GMP binding proteins utilizing a biotinylated cyclic di-GMP pull-down assay. In Methods in Molecular Biology (Vol. 1657, pp. 317–329). Humana Press Inc. https://doi.org/10.1007/978-1-4939-7240-1_25
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