Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pull-Down Assay.
Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pull-Down Assay.
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DOI：
10.1007/978-1-4939-7240-1_25
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Sauer K
中科院分区：
文献类型：
--
作者：
Chambers JR;Sauer K
环二GMP是一种重要的调节信使分子，经常直接与蛋白质相互作用以改变功能。因此，重要的是找到潜在的c-di-GMP结合蛋白，并验证它们之间的直接相互作用。在这里，我们描述了一个下拉测定使用生物素化的-c-二-GMP捕获特定的蛋白质的兴趣，然后通过免疫印迹分析，以确定相对蛋白质丰度。该方法还允许添加特异性和非特异性竞争物，以测定c-di-GMP：蛋白结合的特异性。我们还讨论了使用密度分析所得的免疫印迹计算的结合反应的解离常数（KD），允许结合亲和力的测定。
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 pulldown 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.