Fig. 6.

Evaluation of the dual-readout F² assay performance in an uHTS format. Eighty 1,536-well plates each containing 32 free tracer control wells (125 nM TMR-Noxa peptide) and 32 bound tracer control wells (125 nM TMR-Noxa and 62.5 nM Mcl-1) in a total of 5 (L assay buffer per well were used to determine the assay performance for uHTS. (A) In the TR-FRET readout, the TR-FRET signal for each well was recorded; the average values from each plate were plotted against the corresponding plate number. (B) In the FP readout, the FP signal for each well was measured and the average FP signals from free and bound tracer were plotted against plate number. (C) The S/B values of TR-FRET readout and the S/N values of FP readout were calculated for each plate. (D) Z′ factors for each plate were calculated for both TR-FRET and FP readout. uHTS, ultra-high-throughput screening.