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A simple in vitro method to measure autophosphorylation of protein kinases

Overview of attention for article published in Plant Methods, June 2013
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Title
A simple in vitro method to measure autophosphorylation of protein kinases
Published in
Plant Methods, June 2013
DOI 10.1186/1746-4811-9-22
Pubmed ID
Abstract

Receptor-like protein kinases (RLKs) are a large and important group of plant proteins involved in numerous aspects of development and stress response. Within this family, homo-oligermization of receptors followed by autophosphorylation of the intracellular protein kinase domain appears to be a widespread mechanism to regulate protein kinase activity. In vitro studies of several RLKs have identified autophosphorylation sites involved in regulation of catalytic activity and signaling in vivo. Recent work has established that multiple RLKs are biochemically active when expressed in E. coli and readily autophosphorylate prior to purification or subsequent manipulation. This observation has led us to develop a simplified method for assaying RLK phosphorylation status as an indirect measure of intrinsic autophosphorylation activity. The method involves expressing a recombinant RLK protein kinase domain in E. coli, followed by SDS-PAGE of boiled cell lysate, and sequential staining with the phosphoprotein stain Pro-Q Diamond and a colloidal Coomassie total protein stain. We show this method can be used to measure and quantify in vitro autophosphorylation levels of recombinant wildtype and mutant versions of the Arabidopsis RLK HAESA, as well as to detect transphosphorylation activity of recombinant HAESA against a protein kinase inactive version of itself. Our method has several advantages over traditional protein kinase assays. It does not require protein purification, transfer, blotting, or radioactive reagents. It allows for rapid and quantitative assessment of autophosphorylation levels and should have general utility in the study of any autophosphorylating protein kinase expressed in E. coli.

Twitter Demographics

The data shown below were collected from the profiles of 2 tweeters who shared this research output. Click here to find out more about how the information was compiled.

Mendeley readers

The data shown below were compiled from readership statistics for 62 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Germany 2 3%
Italy 1 2%
Taiwan 1 2%
Unknown 58 94%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 23 37%
Researcher 11 18%
Student > Bachelor 6 10%
Student > Master 6 10%
Student > Doctoral Student 5 8%
Other 7 11%
Unknown 4 6%
Readers by discipline Count As %
Agricultural and Biological Sciences 36 58%
Biochemistry, Genetics and Molecular Biology 12 19%
Engineering 2 3%
Unspecified 1 2%
Computer Science 1 2%
Other 5 8%
Unknown 5 8%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 27 June 2013.
All research outputs
#2,305,585
of 4,507,280 outputs
Outputs from Plant Methods
#107
of 171 outputs
Outputs of similar age
#45,079
of 89,598 outputs
Outputs of similar age from Plant Methods
#11
of 14 outputs
Altmetric has tracked 4,507,280 research outputs across all sources so far. This one is in the 35th percentile – i.e., 35% of other outputs scored the same or lower than it.
So far Altmetric has tracked 171 research outputs from this source. They receive a mean Attention Score of 4.4. This one is in the 21st percentile – i.e., 21% of its peers scored the same or lower than it.
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We're also able to compare this research output to 14 others from the same source and published within six weeks on either side of this one. This one is in the 14th percentile – i.e., 14% of its contemporaries scored the same or lower than it.