Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pull-Down Assay. 2017

Jacob R Chambers, and Karin Sauer
Binghamton Biofilm Research Center (BBRC), Department of Biological Sciences, Binghamton University, 2401 ITC Building, 85 Murray Hill Road, Binghamton, NY, 13902, USA.

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.

UI MeSH Term Description Entries
D007697 Kinesics Systematic study of the body and the use of its static and dynamic position as a means of communication. Body Language,Body Languages,Language, Body,Languages, Body
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D003720 Densitometry The measurement of the density of a material by measuring the amount of light or radiation passing through (or absorbed by) the material. Densitometries
D004268 DNA-Binding Proteins Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases. DNA Helix Destabilizing Proteins,DNA-Binding Protein,Single-Stranded DNA Binding Proteins,DNA Binding Protein,DNA Single-Stranded Binding Protein,SS DNA BP,Single-Stranded DNA-Binding Protein,Binding Protein, DNA,DNA Binding Proteins,DNA Single Stranded Binding Protein,DNA-Binding Protein, Single-Stranded,Protein, DNA-Binding,Single Stranded DNA Binding Protein,Single Stranded DNA Binding Proteins
D006152 Cyclic GMP Guanosine cyclic 3',5'-(hydrogen phosphate). A guanine nucleotide containing one phosphate group which is esterified to the sugar moiety in both the 3'- and 5'-positions. It is a cellular regulatory agent and has been described as a second messenger. Its levels increase in response to a variety of hormones, including acetylcholine, insulin, and oxytocin and it has been found to activate specific protein kinases. (From Merck Index, 11th ed) Guanosine Cyclic 3',5'-Monophosphate,Guanosine Cyclic 3,5 Monophosphate,Guanosine Cyclic Monophosphate,Guanosine Cyclic-3',5'-Monophosphate,3',5'-Monophosphate, Guanosine Cyclic,Cyclic 3',5'-Monophosphate, Guanosine,Cyclic Monophosphate, Guanosine,Cyclic-3',5'-Monophosphate, Guanosine,GMP, Cyclic,Guanosine Cyclic 3',5' Monophosphate,Monophosphate, Guanosine Cyclic
D015151 Immunoblotting Immunologic method used for detecting or quantifying immunoreactive substances. The substance is identified by first immobilizing it by blotting onto a membrane and then tagging it with labeled antibodies. Dot Immunoblotting,Electroimmunoblotting,Immunoelectroblotting,Reverse Immunoblotting,Immunoblotting, Dot,Immunoblotting, Reverse,Dot Immunoblottings,Electroimmunoblottings,Immunoblottings,Immunoblottings, Dot,Immunoblottings, Reverse,Immunoelectroblottings,Reverse Immunoblottings
D019921 Biotinylation Incorporation of biotinyl groups into molecules. Biotinylations

Related Publications

Jacob R Chambers, and Karin Sauer
January 2015, Bio-protocol,
Jacob R Chambers, and Karin Sauer
January 2016, Journal of bacteriology,
Jacob R Chambers, and Karin Sauer
February 2022, Bio-protocol,
Jacob R Chambers, and Karin Sauer
January 2021, Methods in molecular biology (Clifton, N.J.),
Jacob R Chambers, and Karin Sauer
February 2023, Journal of visualized experiments : JoVE,
Jacob R Chambers, and Karin Sauer
May 2017, Bio-protocol,
Jacob R Chambers, and Karin Sauer
January 2012, Journal of visualized experiments : JoVE,
Jacob R Chambers, and Karin Sauer
August 2012, Journal of proteomics,
Copied contents to your clipboard!