Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
KBI Biopharma, Inc., Boulder, CO, USA.
Methods Mol Biol. 2024;2743:285-300. doi: 10.1007/978-1-0716-3569-8_18.
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.
蛋白酪氨酸磷酸酶（PTP），如 Eyes Absent（Eya）家族蛋白，在多种生物过程中发挥重要作用。体外磷酸酶检测是表征酶活性以及发现这些磷酸酶抑制剂和调节剂的重要工具。两种常见的体外磷酸酶检测方法使用小分子底物产生荧光或有色产物，或使用肽底物在孔雀绿检测中产生比色产物。在本章中，我们描述了使用小分子 3-O-甲基荧光素磷酸酯（OMFP）作为底物的磷酸酶检测以及使用 pH2AX 肽作为底物的孔雀绿检测的详细方案，以评估 EYA2 的磷酸酶活性和 EYA2 的小分子抑制剂的影响。这些方案可以很容易地适应于研究其他蛋白酪氨酸磷酸酶。
