Sufficient ionic strength from KCl ensured reliable Cytochrome C and Smac release from mitochondria permeabilized by BH3 peptides. 2LMP mitochondria were isolated in mitochondrial isolation buffer (MIB pH 7.4) containing 10 mM Tris–HCl, 0.1 mM EDTA, 250 mM Sucrose and permeabilized by Bim BH3 peptides (26-mer unless otherwise indicated) for 60 min in mitochondrial reaction buffer (MRB pH 7.4) containing 20 mM HEPES 1.5 mM MgCl2, 1 mM EDTA, 250 mM sucrose, and different concentrations of KCl (A). The ionic strength-dependent release of Cytochrome C and Smac proteins was confirmed by densitometry analysis (A). 2LMP mitochondria were permeabilized by Bim BH3 at indicated concentrations for 30 min or 60 min in either high ionic strength buffer containing 100 mM KCl or low ionic strength buffer containing 10 mM KCl (B). Mitochondrial pellets (P) and supernatants (S) were carefully separated and mixed with sample buffer, and equivalent portions were subjected to SDS-PAGE and Western blotting analysis for Cytochrome C and Smac/Diablo. Results are representative of three independent experiments. *: 21-mer Bim BH3 peptide.