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Global identification of protein kinase substrates by protein microarray analysis

Nature Protocols volume 4, pages 1820–1827 (2009)Cite this article

Abstract

Herein, we describe a protocol for the global identification of in vitro substrates targeted by protein kinases using protein microarray technology. Large numbers of fusion proteins tagged at their carboxy-termini are purified in 96-well format and spotted in duplicate onto amino-silane-coated slides in a spatially addressable manner. These arrays are incubated in the presence of purified kinase and radiolabeled ATP, and then washed, dried and analyzed by autoradiography. The extent of phosphorylation of each spot is quantified and normalized, and proteins that are reproducibly phosphorylated in the presence of the active kinase relative to control slides are scored as positive substrates. This approach enables the rapid determination of kinase–substrate relationship on a proteome-wide scale, and although developed using yeast, has since been adapted to higher eukaryotic systems. Expression, purification and printing of the yeast proteome require about 3 weeks. Afterwards, each kinase assay takes ∼3 h to perform.

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Figure 1: Workflow of a protein microarray-based kinase assay to identify candidate substrates targeted by the yeast kinases.

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Acknowledgements

This work was supported by a grant from the NIH. The assays shown in Figure 1 were performed by Dr. Jason Ptacek and Dr. Geeta Devgan. We thank Dr. Rui Chen and Dr. Vincent Bruno for helpful comments.

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Author notes

  1. Michael Snyder

    Present address: Present addresses: Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA (J.M.); Department of Genetics, Stanford University, Palo Alto, California, USA (H.I. and M.S.).,

  2. Janine Mok and Hogune Im: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA

    Janine Mok, Hogune Im & Michael Snyder

Authors
  1. Janine Mok
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  2. Hogune Im
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  3. Michael Snyder
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All authors contributed extensively to the work presented in this paper.

Corresponding author

Correspondence to Michael Snyder.

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Mok, J., Im, H. & Snyder, M. Global identification of protein kinase substrates by protein microarray analysis. Nat Protoc 4, 1820–1827 (2009). https://doi.org/10.1038/nprot.2009.194

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