Immunoblotting of Superoxide Dismutase 1

Crude homogenates were diluted 1:10 in 1 × SDS sample buffer without prior centrifugation and heated for 10 min at 100 °C before separation on Any kD Criterion TGX precast gels (Bio-Rad). Either anti-hSOD1 mAbs (1 μg/mL), mouse anti-c-MYC IgG1 (1 μg/mL) or mouse anti-β-actin (1:100,000; Merck Millipore) were used as primary antibodies and HRP-conjugated anti-mouse IgG (1:10,000, DAKO) as a secondary antibody. Immunocaptured samples were analysed using rabbit polyclonal antibodies generated against a peptide corresponding to amino acids a.a. 24–39 in the hSOD1 sequence [15 (link)] and an anti-rabbit IgG (1:10,000, DAKO) as secondary antibody. The immunoreaction signal was visualized using ECL Select reagent (GE Healthcare), recorded on a ChemiDoc Touch Imaging system and analysed using Image Lab software (Bio-Rad).