Prism software 5

For direct FP binding assays, FlTC-derivatized BIM BH3 peptides (50 nM) were added to serial dilutions of recombinant protein in binding buffer (BCL-X_L ΔC: 100 mM NaCl, 50 mM Tris, pH 8.0; full-length BAX and BCL-X_L: 140 mM NaCl, 50 mM Tris, pH 7.4) in 96-well black opaque plates. For binding assays employing BAX A112C/V177C, the protein stock and binding buffer was supplemented with GSSG (0.5 mM). The plates were incubated in the dark at RT and then fluorescence polarization measured at 20 min on a microplate reader (SpectraMax M5 Microplate Reader, Molecular Devices). Dissociation constants (K_D) were calculated by nonlinear regression analysis of dose-response curves with Prism software 5.0 (GraphPad) using the ligand depletion formula for FP binding assays, as described.^{13 (link),35} For competitive FP binding assays, FITC-derivatized BID BH3 peptide (25 nM) composed of the sequence DIIRNIARHLAQVGDSBDRSI (where B is norleucine) was added to BCL-X_L ΔC protein (250 nM) and serial dilutions (starting from 1 μm) of Ac-BIM BH3₁ or Ac-BIM SAHB_A1 in 96-well black opaque plates. The plates were incubated in the dark at RT and then FP measured at 20 min as above. IC50s were calculated by nonlinear regression analysis of dose-response curves using Prism software 5.0 (GraphPad) and Kis calculated according to the formula, Ki = IC50/(1 + [L/K_D]).