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Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pull-Down Assay

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c-di-GMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1657))

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.

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Acknowledgments

This work was supported by a grant from the National Institute of Health (R01AI080710).

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Correspondence to Karin Sauer .

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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

  • Print ISBN: 978-1-4939-7239-5

  • Online ISBN: 978-1-4939-7240-1

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