Polyvinyl difluoride pvdf membranes

To obtain mitochondria-enriched fractions, adrenal glands were homogenized in 4 volumes of 300 mmol/L sucrose with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 1 μg/ml aprotinin). Homogenates were centrifuged at 1000g to remove unbroken cells, nuclei and heavy membranes. Mitochondria-enriched fractions were obtained by centrifugation at 3000g at 4 C for 15min. Then, it was further centrifuged at 45000g for 1 h and the obtained supernatant was used as the cytosolic fraction, as previously reported (Ronco et al., 2004) (link).
Proteins were quantified according to Lowry technique. For protein detection were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro blotted onto polyvinyl difluoride (PVDF) membranes (PerkinElmer Life Sciences, Inc., Boston, MA).
Membranes were incubated with primary anti-mouse antibodies (anti-Bax, anti-Bid, anti-XIAP;
anti-cytochrome c, all from Santa Cruz Biotechnology). Finally, protein levels were detected by enhanced chemiluminescence detection system (Pierce ECL, Thermo Fisher Scientific).
Immunoreactive bands were quantified by densitometry using the Image J software (imagej.nih.gov).