Experimental and Analytical Approaches to Characterize Plant Kinases Using Protein Microarrays
Comprehensive analysis of protein kinases and cellular signaling pathways requires the identification of kinase substrates and interaction partners using large-scale amenable approaches. Here, we describe our methods for producing plant protein microarrays (PMAs) and discuss various parameters critical to the quality of PMAs. Next, we describe methods for detecting protein-protein interactions and kinase activity including auto-phosphorylation and substrate phosphorylation. We have provided a short video demonstrating how to conduct an interaction assay and how to properly handle a protein microarray. Finally, a set of analytical methods are presented as a bioinformatics pipeline for the acquisition of PMA data and for selecting PMA candidates using statistical testing. The experimental and analytical protocols described here outline the steps to produce and utilize PMAs to analyze signaling networks.
Key wordsProtein microarrays Kinase substrates Protein interaction Phosphorylation assays Statistical decision
We are grateful to Claire Smith and Kent Loeffler from the Photo Laboratory in the Department of Plant Pathology and Plant Microbe Interactions (Cornell University) for the help with producing the movie accompanying this manuscript. This work was supported by the National Science and Engineering Research Council of Canada (post-graduate fellowship to E. K. Brauer), the National Science Foundation (project IOS-1025642 to S. C. Popescu) and National Research Council-Executive Agency for Higher Education, Research, Development and Innovation Funding (projects PN-II-PT-PCCA-2011-3.1-1350 and PN-II-CT-RO-FR-2012-1-709 to G. V. Popescu).
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