Figure 2

TR-FRET assay characterization for reliability, robustness and reproducibility. (A) Dynamic range of both time-resolved Förster’s resonance energy transfer (TR-FRET) assays was assessed by serial dilutions of purified recombinant FMRP starting at 2,000 pg/μl into assay buffer in wells of low-volume 384-well plates. (B) Limits of detection for both assays were determined according to the standards set by the Clinical and Laboratory Standards Institute[12]. The TR-FRET signal intensity for FMRP protein concentrations around the expected putative limits of detection (as previously examined in the dynamic range assessment) were compared with the limit of blank for each assay. Limit of detection was defined as the FMRP concentration at which ≥95% of analyzed samples resulted in a TR-FRET signal above the limit of blank. Determined limit of detection concentrations for each assay are indicated by grey symbols. (C) Assessment of intra-assay variability for each assay with three different FMRP concentrations distributed randomly across a low-volume 384-well plate (locations I to III). (D) Assessment of inter-assay variability was determined for each assay by testing reused frozen and thawed protein standard and antibodies (a), reused protein standard but fresh antibody detection solution (b) or freshly diluted protein standard and antibody solution (c) on two independent days. All values for A, C and D are presented as percentage signal over assay buffer background. All data and error bars represent averages and standard deviations of triplicates.