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Optimization of the 14-3-3/pBad peptide binding assay in a 384-well HTS format. (A) The TR-FRET signal increased by increasing concentrations of His-14-3-3ζ protein. His-14-3-3ζ protein was titrated against various concentrations of the Dy647-pS136-Bad peptide in the presence of anti–His-Eu antibody (1 nM). The S/B (B) and Z′ factor (C) were calculated and plotted against His-14-3-3ζ protein concentrations.
