An affinity pull-down approach to identify the plant cyclic nucleotide interactome

Handle URI:
http://hdl.handle.net/10754/562961
Title:
An affinity pull-down approach to identify the plant cyclic nucleotide interactome
Authors:
Donaldson, Lara Elizabeth; Meier, Stuart Kurt
Abstract:
Cyclic 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.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Publisher:
Humana Press
Journal:
Methods in Molecular Biology
Issue Date:
3-Sep-2013
DOI:
10.1007/978-1-62703-441-8-11
PubMed ID:
23681578
Type:
Article
ISSN:
10643745
ISBN:
9781627034401
Appears in Collections:
Articles; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorDonaldson, Lara Elizabethen
dc.contributor.authorMeier, Stuart Kurten
dc.date.accessioned2015-08-03T11:17:09Zen
dc.date.available2015-08-03T11:17:09Zen
dc.date.issued2013-09-03en
dc.identifier.isbn9781627034401en
dc.identifier.issn10643745en
dc.identifier.pmid23681578en
dc.identifier.doi10.1007/978-1-62703-441-8-11en
dc.identifier.urihttp://hdl.handle.net/10754/562961en
dc.description.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.en
dc.publisherHumana Pressen
dc.subjectAdenosine 3′, 5′-cyclic monophosphate (cAMP)en
dc.subjectCyclic nucleotideen
dc.subjectCyclic nucleotide-binding domainen
dc.subjectCyclic nucleotide-binding proteinen
dc.subjectGuanosine 3′5′-cyclic monophosphate (cGMP)en
dc.titleAn affinity pull-down approach to identify the plant cyclic nucleotide interactomeen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.identifier.journalMethods in Molecular Biologyen
kaust.authorDonaldson, Lara Elizabethen
kaust.authorMeier, Stuart Kurten

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