2.4. Determination of Antioxidant Enzymes by Western Blot

Determination of EADS enzymes was conducted by means of the Western blot technique, in which the enzymes were submitted to electrophoresis (Mini Protean 3 Electrophoresis System and, as power source, Power Pac High-Current (all these purchased from Bio-Rad, Hercules, CA, USA)), in acrylamide gel, and later immunotransference based on the specificity of antibody–antigen recognition. Equal amounts of protein lysates obtained from rat-liver homogenates were separated in 10% sodium dodecyl sulfate–polyacrylamide (SDS–PAGE) gel electrophoresis. The gels were blotted onto a polyvinylidene difluoride (PVDF) Amersham Hybond-P membrane (GE Healthcare, Buckinghamshire, UK) and incubated with the appropriate antibodies: antisuperoxide dismutase (S2147; 1:2000 dilution), anticatalase (C0979; 1:3000 dilution), anti-β-actin (A2228; 1:50,000 dilution) from Sigma-Aldrich, St. Louis, MO, USA; antiglutathione peroxidase (Ab59546; 1:5000 dilution) from Abcam, Cambridge, UK; antiglutathione reductase (sc-32886; 1:500 dilution), mouse antigoat (sc-2490; 1:20,000 dilution) and goat antirabbit (sc-2004; 1:10,000 dilution) from Santa Cruz Biotechnology, Dallas, TX, USA). Blots were developed by enhanced chemoluminescence using an Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. β-actin was used as a loading control [10].