Preparation of Recombinant Protein Spotted Arrays for Proteome‐Wide Identification of Kinase Targets
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- Abstract
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Abstract
Protein microarrays allow unique approaches for interrogating global protein interaction networks. Protein arrays can be divided into two categories: antibody arrays and functional protein arrays. Antibody arrays consist of various antibodies and are appropriate for profiling protein abundance and modifications. Functional full?length protein arrays employ full?length proteins with various post?translational modifications. A key advantage of the latter is rapid parallel processing of large number of proteins for studying highly controlled biochemical activities, protein?protein interactions, protein?nucleic acid interactions, and protein?small molecule interactions. This unit presents a protocol for constructing functional yeast protein microarrays for global kinase substrate identification. This approach enables the rapid determination of protein interaction networks in yeast on a proteome?wide level. The same methodology can be readily applied to higher eukaryotic systems with careful consideration of overexpression strategy. Curr. Protoc. Protein Sci. 72:27.4.1?27.4.14. © 2013 by John Wiley & Sons, Inc.
Keywords: protein array; post?translation; phosphorylation; kinase
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Strategic Planning
- Basic Protocol 1: Protein Induction and Purification of Proteins for Printing
- Basic Protocol 2: Printing of Proteome Microarrays
- Basic Protocol 3: Perform Radioactive In Vitro Kinase Assay on a Protein Microarray
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Protein Induction and Purification of Proteins for Printing
Materials
Basic Protocol 2: Printing of Proteome Microarrays
Materials
Basic Protocol 3: Perform Radioactive In Vitro Kinase Assay on a Protein Microarray
Materials
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Figure 27.4.1 Schematic diagram of overall protocol. Yeast clones containing cDNA constructs are plated on selection medium. Each clone is grown and protein expression is induced. Pellets of each clone are transferred into 96‐well boxes. Fusion proteins are purified and transferred to 96‐well titer plates for printing on slides. In the upper right, the kinase‐treated block shows spots, which indicate phosphorylation of printed proteins with radioactive phosphate. Spots in the corner rectangles are positive controls. View Image
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Literature Cited
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