2D Western blot of superoxide dismutase (Mn-SOD) in isolated mitochondria

Mitochondria were isolated from NIH3T3 cells using the Dounce homogenization protocol and re-suspended in 2D sample buffer (7M urea, 2M thiourea, 4% CHAPS, pH 3-10 carrier ampholytes, 50mM DTT). Proteins were resolved on a pH 3-10 IPG strip followed by 8-16% SDS-PAGE and then analyzed by Western blot for superoxide dismutase.