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. 2013 Jul;11(6):367–381. doi: 10.1089/adt.2013.507

Fig. 2.

Fig. 2.

Evaluation of the Dy647-pS136-Bad peptide in the 14-3-3 TR-FRET assay in a 384-well high-throughput screening (HTS) format. (A) Increasing concentrations of Dy647-pS136-Bad peptide (15 μL) were mixed with the indicated concentrations of His-14-3-3ζ protein (15 μL) in the presence of anti–His-Eu antibody (1 nM, final). TR-FRET signals were recorded using an EnVision multilabel plate reader. The FRET signal window was calculated by subtracting the background signal from the Dy647-pS136-Bad peptide and anti–His-Eu only from the FRET signal obtained in the presence of 14-3-3 protein, the Dy647-pS136-Bad peptide, and anti–His-Eu (A). (B, C) The performance of the assay for HTS was evaluated by the Z′ factor and signal-to-background ratio (S/B). S/B (B) and Z′ (C) were calculated and plotted against Dy647-pS136 peptide concentrations.