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In Vitro Phosphatase Assays for the Eya2 Tyrosine Phosphatase

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Protein Tyrosine Phosphatases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2743))

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Abstract

Protein tyrosine phosphatases (PTP), such as the Eyes Absent (Eya) family of proteins, play important roles in diverse biological processes. In vitro phosphatase assays are essential tools for characterizing the enzymatic activity as well as discovering inhibitors and regulators of these phosphatases. Two common types of in vitro phosphatase assays use either a small molecule substrate that produces a fluorescent or colored product, or a peptide substrate that produces a colorimetric product in a malachite green assay. In this chapter, we describe detailed protocols of a phosphatase assay using small molecule 3-O-methylfluorescein phosphate (OMFP) as a substrate and a malachite green assay using the pH2AX peptide as a substrate to evaluate the phosphatase activity of EYA2 and the effect of small molecule inhibitors of EYA2. These protocols can be easily adapted to study other protein tyrosine phosphatases.

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Acknowledgments

Research reported in this chapter was supported by the National Cancer Institute of the National Institute of Health under award number R03DA030559 (R.Z. and H.L.F.), R41CA180347 (H.L.F. and R.Z.), R01CA221282 (H.L.F. and R.Z.), R01CA224867 (H.L.F.), R01NS108396 (H.L.F.), Colorado Bioscience Discovery and Evaluation Grant (H.L.F. and R.Z.), and Cancer League of Colorado Grant (H.L.F. and R.Z.), as well as National Institute of General Medical Sciences of the National Institute of Health under award number R35GM145289 (R.Z.). C.A. is supported by the NIH NRSA T32CA174648 Training in Translational Research of Lung, Head and Neck Cancer. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Christopher Alderman, Aaron Krueger, John Rossi & Rui Zhao

  2. KBI Biopharma, Inc., Boulder, CO, USA

    Aaron Krueger

  3. Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Heide L. Ford

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  1. Christopher Alderman
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  2. Aaron Krueger
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  3. John Rossi
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  4. Heide L. Ford
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  5. Rui Zhao
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Corresponding author

Correspondence to Rui Zhao .

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Editors and Affiliations

  1. Department of Chemistry, Lehigh University, Bethlehem, PA, USA

    Damien Thévenin

  2. Center for Molecular Biomedicine, University Hospital, Friedrich Schiller University Jena, Jena, Germany

    Jörg P. Müller

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Alderman, C., Krueger, A., Rossi, J., Ford, H.L., Zhao, R. (2024). In Vitro Phosphatase Assays for the Eya2 Tyrosine Phosphatase. In: Thévenin, D., P. Müller, J. (eds) Protein Tyrosine Phosphatases. Methods in Molecular Biology, vol 2743. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3569-8_18

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  • DOI: https://doi.org/10.1007/978-1-0716-3569-8_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3568-1

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