Mouse monoclonal anti β actin clone ac 15

For protein analysis by western blot techniques, samples from HeLa controls, ZnPc-PDT, TMPyP-PDT, and ZnPc+TMPyP-PDT-treated cell cultures were processed 6 h after the corresponding treatment. Whole protein content was extracted by lysis using RIPA buffer (50 ml of distilled water with 50 mM Tris-HCl at pH 8; 150 mM NaCl; 1% (v/v) Igepal CA630 (all from Sigma-Aldrich) and 0.1% (v/v) SDS; complemented with a tablet of complete EDTA-free protease inhibitors (Roche)). Protein concentration was determined using a BCA assay kit (Pierce, Rockford, IL, USA), and samples were loaded and separated on 15% SDS-PAGE and transferred to a nitrocellulose membrane using a Mini-Protean 3 equipment, according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). Chemoluminescent detection of proteins was performed with a mouse monoclonal anti-β-actin (clone AC-15; Sigma-Aldrich) antibody, and a mouse monoclonal anti-PARP (clone C-2-10; Sigma-Aldrich) antibody, using overnight incubation at 4 °C. Secondary antibody was sheep anti-mouse IgG conjugated to horseradish peroxidase (Amersham Biosciences, Buckinghamshire, UK). Bands were developed on a Curix CP-G Plus paper (AGFA, Barcelona, Spain) with Western Blotting Luminol Reagent (Roche).