Fig. (6).

Evaluation of the DMSO tolerance and stability of the 14-3-3 AlphaScreen assay. (A) Effect of DMSO on Alpha signal. The biotin-14-3-3γ (5 nM) and GST-R18 (15 nM) were incubated with increasing amounts of DMSO, and reactions were carried out as described in the legend to Fig. (4). Results were recorded after incubation with donor and acceptor beads for 1.5 hr. (B) Stability of the 14-3-3 AlphaScreen assay as evaluated in R18 competition assay. Experiments were carried out as described in Fig. (4) except that results were recorded after incubation for 1.5 hr or 16 hr. Dose-response data with the untagged R18 peptide antagonist were expressed as the percentage of maximal Alpha signal control that was obtained from samples in the absence of inhibitor. Data are expressed as means ± SD from triplicate determinations.