In Vitro Phosphatase Assays for the Eya2 Tyrosine Phosphatase.
In Vitro Phosphatase Assays for the Eya2 Tyrosine Phosphatase.
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Eya2 酪氨酸磷酸酶的体外磷酸酶测定。
DOI：
10.1007/978-1-0716-3569-8_18
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Zhao,Rui
中科院分区：
文献类型：
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作者：
Alderman,Christopher;Krueger,Aaron;Rossi,John;Ford,HeideL;Zhao,Rui
蛋白酪氨酸磷酸酶(PTP)，如眼睛缺失(EYA)蛋白家族，在多种生物过程中发挥着重要作用。体外磷酸酶检测是表征酶活性以及发现这些磷酸酶的抑制剂和调节剂的重要工具。两种常见类型的体外磷酸酶检测要么使用产生荧光或有色产物的小分子底物，要么使用在孔雀石绿测定中产生比色产物的多肽底物。在本章中，我们描述了以小分子3-O-甲基荧光素磷酸(OMFP)为底物的磷酸酶检测方法和以pH2AX肽为底物的孔雀绿检测方法，以评估EYA2的磷酸酶活性以及EYA2的小分子抑制剂的影响。这些方案可以很容易地适用于研究其他蛋白质酪氨酸磷酸酶。
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.