Fig. (3).

AlphaScreen assay for measuring the interaction of 14-3-3γ with R18. (A) Biotinylation has no effect on the ligand binding capability of His-14-3-3γ proteins. Binding of biotin-14-3-3γ to pS259-Raf-1 peptide was determined by FP assay [11]. Biotin-14-3-3 proteins were incubated with a TMR-pS259-Raf peptide (1 nM) at RT for 30 min and the FP signal [expressed in millipolarization units (mP)] was read with an Analyst HT reader (Molecular Devices). Specific binding (mP) was obtained by subtracting free peptide tracer values from values recorded in the presence of 14-3-3 proteins. (B) Interaction of biotin-14-3-3 with GST-R18 generates an Alpha luminescence signal. Increasing concentrations of GST-R18 protein were incubated with biotin-14-3-3 protein at RT for 30 min. Donor and acceptor beads were added with an interval of 30 min. The plate was assayed using an EnVision multilabel plate reader after 1.5 hr incubation at room temperature. Data are expressed as mean ± SD from triplicate samples. (C) S/B ratios of the 14-3-3γ/R18 AlphaScreen assay. (D) Z’ values of the assay.