Plant MAP Kinases pp 217-235

Part of the Methods in Molecular Biology book series (MIMB, volume 1171) | Cite as

Experimental and Analytical Approaches to Characterize Plant Kinases Using Protein Microarrays

Protocol

Abstract

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 words

Protein microarrays Kinase substrates Protein interaction Phosphorylation assays Statistical decision 

Supplementary material

(MOV 766212 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.The Boyce Thompson Institute for Plant ResearchIthacaUSA
  2. 2.Department of Plant Pathology and Plant Microbe BiologyCornell UniversityIthacaUSA
  3. 3.National Institute for Laser, Plasma & Radiation PhysicsBucharestRomania

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