Chapter title |
Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pull-Down Assay.
|
---|---|
Chapter number | 25 |
Book title |
c-di-GMP Signaling
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7240-1_25 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7239-5, 978-1-4939-7240-1
|
Authors |
Chambers, Jacob R, Sauer, Karin, Jacob R. Chambers, Karin Sauer |
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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