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Updated: Jul 7, 2026
Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells
Published on: September 9, 2021
A high-throughput assay for signal transducer and activator of transcription 5b based on fluorescence polarization
Judith Müller1, Jochen Schust, Thorsten Berg
1Max Planck Institute of Biochemistry, Department of Molecular Biology, and Munich Center for Integrated Protein Science (CIPSM), Am Klopferspitz 18, 82152 Martinsried, Germany.
Abstract:
Signal transducer and activator of transcription 5b (STAT5b) is constitutively activated in many human tumors. Activity of STAT5b requires binding of its Src homology 2 (SH2) domain to certain phosphotyrosine-containing sequences. We have developed a high-throughput assay based on fluorescence polarization that allows screening of chemical libraries for compounds that inhibit STAT5b by interfering with the function of its SH2 domain. The assay, which is based on binding between a fluorescein-labeled phosphotyrosine peptide derived from the erythropoietin receptor to the STAT5b SH2 domain, is stable with regard to dimethyl sulfoxide concentration and time and has a Z' value of 0.66+/-0.11 in a 384-well format.
