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Methods Mol Biol. 2016;1352:27-34. doi: 10.1007/978-1-4939-3037-1_3.

Analysis of Protein Tyrosine Kinase Specificity Using Positional Scanning Peptide Microarrays.

Author information

1
Department of Biomedical Engineering, Yale University, New Haven, CT, USA.
2
Department of Pharmacology, Yale University School of Medicine, SHM B395e, 333 Cedar Street, New Haven, CT, 06520, USA. ben.turk@yale.edu.

Abstract

Protein tyrosine kinases phosphorylate their substrates within the context of specific consensus sequences surrounding the site of modification. We describe a peptide microarray approach to rapidly determine tyrosine kinase phosphorylation site motifs. This method uses a peptide library that systematically substitutes each of the amino acid residues at multiple positions surrounding a central tyrosine residue. Peptide substrates are synthesized as biotin conjugates for immobilization on avidin-coated slides. Following incubation of the slide with protein kinase and radiolabeled ATP, the relative extent of phosphorylation of each of the peptides is quantified by phosphor imaging. This method allows small quantities of kinase to be analyzed rapidly in parallel, facilitating analysis of large numbers of kinases.

KEYWORDS:

Enzyme specificity; Kinase assay; Peptide microarrays; Protein tyrosine kinase s; Signal transduction; Substrate profiling

PMID:
26490465
PMCID:
PMC4814292
DOI:
10.1007/978-1-4939-3037-1_3
[Indexed for MEDLINE]
Free PMC Article

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