Mouse monoclonal anti cytochrome c antibody

Mitochondria (60 μg in protein quantity) were spun down at 12,000 g for 10 min at 4 °C. They were resuspended in 100 μl of the cytochrome c release assay buffer (20 mM HEPES/KOH pH 7.5, 100 mM sucrose, 80 mM KCl, 1 mM ATP, 80 μM ADP, 5 mM Na Succinate, 1 mM DL-dithiothreitol (DTT)) in the presence of 0 or 100 nM p7/p15 Bid, and further incubated for 30 min at 30 °C. A volume of 50 μl of the reaction mixture was set aside on ice for the cross-linking experiments below. Cytochrome c released into the medium was collected by centrifuging the remaining samples at 12,000 g for 10 min at 4 °C. The resulting pellet was resupended in the assay buffer (50 μl). A volume of 10 μl of 6x SDS sample buffer (0.375 M Tris pH 6.8, 12% (w/v) SDS, 60% (v/v) glycerol, 0.6 M DTT, 0.06% (w/v) bromophenol blue) was mixed with 50 μl of the resulting supernatant and resuspended mitochondrial samples. One sixth of each paired sample was subjected to SDS-PAGE under a reducing condition, followed by immunoblotting. The primary and the secondary antibodies used were mouse monoclonal anti-cytochrome c antibody (Santa Cruz, Cat. # sc-13156)/Anti-rabbit IgG (Perkin Elmer, Cat. # NEF812001EA). The percentage of released cytochrome c was determined by measuring the intensities of the Western blotting images using ImageJ software.

The cells were seeded onto two-well chamber slides and transfected for 72 h with TFF3-siRNA and siCtrl. After blocking with 1% BSA in TBS, the cells were incubated overnight at 4 ℃ with primary antibodies, including a rabbit polyclonal anti-TFF3 antibody (Santa Cruz), a rabbit monoclonal anti-cleaved caspase-3 antibody (Cell Signaling Technology), a mouse monoclonal anti-cytochrome c antibody (Santa Cruz) and a goat polyclonal anti-Smac antibody (Santa Cruz). The slides were rinsed with TBS and then incubated for 1 h with anti-rabbit IgG-FITC (Sigma), anti-mouse IgG-FITC (Santa Cruz) and anti-goat IgG-FITC (Santa Cruz). Then, the cell nuclei were stained with Hoechst 33342 (Life Technologies, Carlsbad, CA), and the mitochondria were stained with the Mito-ID Red detection kit (Enzo Life Sciences, Farmingdale, NY). The slides were analyzed by fluorescence microscope (Axio Imager M1, Carl Zeiss, Oberkochen, Germany). Three fields were randomly selected from three independent experiments.