Protocol abstract


Nature Protocols 3, 1485 - 1493 (2008)
Published online: 28 August 2008 | doi:10.1038/nprot.2008.139

Subject Categories: Chemical modification | Biochemistry and protein analysis

Peptide microarrays for high-throughput studies of Ser/Thr phosphatases

Hongyan Sun1, Candy H S Lu2, Haibin Shi1,3, Liqian Gao1,3 & Shao Q Yao1,2,3


Protein phosphorylation and dephosphorylation play an important role in regulation of intracellular signal transduction pathways in the biological system. A key step in the biological characterization of phosphatases and their use as drug targets is the identification of their cellular partners and suitable substrates for potential inhibitor development. Herein we describe a microarray-based protocol to map the substrate specificity of protein Ser/Thr phosphatases. This protocol uses Pro-Q dye to sensitively and quantitatively detect the amount of dephosphorylation that occurs from many putative peptide substrates in parallel, and therefore could be used to generate the so-called peptide substrate fingerprints as well as detailed kinetic information of a target phosphatase. Excluding the synthesis of the peptide substrates, the whole protocol takes a total of 11 h to complete and in future can be readily extended to the study of other classes of phosphatases, i.e., protein tyrosine phosphatases.

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  1. Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore.
  2. Department of Biological Science, National University of Singapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore.
  3. NUS MedChem Program of the Office of Life Sciences, National University of Singapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore.

Correspondence to: Shao Q Yao1,2,3 e-mail: chmyaosq@nus.edu.sg

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