Abstract
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.
Key words
- Cyclic di-GMP
- Pull-down assay
- Western blot
- Immunoblot
- Biotinylated-c-di-GMP
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Acknowledgments
This work was supported by a grant from the National Institute of Health (R01AI080710).
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Chambers, J.R., Sauer, K. (2017). Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pull-Down Assay. In: Sauer, K. (eds) c-di-GMP Signaling. Methods in Molecular Biology, vol 1657. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7240-1_25
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DOI: https://doi.org/10.1007/978-1-4939-7240-1_25
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7240-1
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