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Development of Fluorescence-Based Selective Assays for Serine / Threonine and Tyrosine Phosphatases

[ Vol. 6 , Issue. 4 ]

Author(s):

Christina Pastula, Iain Johnson, Joseph M. Beechem and Wayne F. Patton   Pages 341 - 346 ( 6 )

Abstract:


A number of aromatic substrates were evaluated for their ability to detect tyrosine phosphatase and serine / threonine phosphatase activity. Results demonstrated that the fluorinated coumarin DiFMUP is the most sensitive substrate for detecting LAR and PP-2A activity. Using this substrate, selective high-throughput screening assays for serine / threonine and tyrosine phosphatases were developed. Specific inhibitor cocktails were added to each assay to limit the activity of other phosphatases. LAR, CD-45, and PTP-1B all rapidly hydrolyze DiFMUP in the tyrosine phosphatase assay. The activity of non-tyrosine phosphatases is less than 6% of the LAR activity. PP-1 and PP-2A are highly active in the serine / threonine phosphatase assay. Inhibition of LAR and PP-2A in these assays is demonstrated using known inhibitors. Both of these assays are sensitive, robust, kinetic assays that can be used to quantify enzyme activity.

Keywords:

fluorescence, microplate, tyrosine phosphatase, serine/threonine phosphatase, phosphatase inhibitor

Affiliation:

Molecular Probes, Inc., Biomics Department, 29851 Willow Creek Rd, Eugene, Oregon, 97402, USA.



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