An affinity pull-down approach to identify the plant cyclic nucleotide interactome
Permanent link to this recordhttp://hdl.handle.net/10754/562961
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AbstractCyclic nucleotides (CNs) are intracellular second messengers that play an important role in mediating physiological responses to environmental and developmental signals, in species ranging from bacteria to humans. In response to these signals, CNs are synthesized by nucleotidyl cyclases and then act by binding to and altering the activity of downstream target proteins known as cyclic nucleotide-binding proteins (CNBPs). A number of CNBPs have been identified across kingdoms including transcription factors, protein kinases, phosphodiesterases, and channels, all of which harbor conserved CN-binding domains. In plants however, few CNBPs have been identified as homology searches fail to return plant sequences with significant matches to known CNBPs. Recently, affinity pull-down techniques have been successfully used to identify CNBPs in animals and have provided new insights into CN signaling. The application of these techniques to plants has not yet been extensively explored and offers an alternative approach toward the unbiased discovery of novel CNBP candidates in plants. Here, an affinity pull-down technique for the identification of the plant CN interactome is presented. In summary, the method involves an extraction of plant proteins which is incubated with a CN-bait, followed by a series of increasingly stringent elutions that eliminates proteins in a sequential manner according to their affinity to the bait. The eluted and bait-bound proteins are separated by one-dimensional gel electrophoresis, excised, and digested with trypsin after which the resultant peptides are identified by mass spectrometry - techniques that are commonplace in proteomics experiments. The discovery of plant CNBPs promises to provide valuable insight into the mechanism of CN signal transduction in plants. © Springer Science+Business Media New York 2013.
JournalMethods in Molecular Biology
- The arabidopsis cyclic nucleotide interactome.
- Authors: Donaldson L, Meier S, Gehring C
- Issue date: 2016 May 11
- Cyclic nucleotides.
- Authors: Newton RP, Smith CJ
- Issue date: 2004 Sep
- Large-scale identification of tubulin-binding proteins provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells.
- Authors: Chuong SD, Good AG, Taylor GJ, Freeman MC, Moorhead GB, Muench DG
- Issue date: 2004 Oct
- Protein complexes characterization in Arabidopsis thaliana by tandem affinity purification coupled to mass spectrometry analysis.
- Authors: Bigeard J, Pflieger D, Colcombet J, Gérard L, Mireau H, Hirt H
- Issue date: 2014
- Detection of reactive oxygen species downstream of cyclic nucleotide signals in plants.
- Authors: Walker RK, Berkowitz GA
- Issue date: 2013
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