The glutathionylated ~ 45 kDa band is sarcomeric mitochondrial creatine kinase, not a complex I subunit. Mitochondria (20 mg protein/ml) were first incubated with [35S]GSH (40 µCi/ml) in STE for 30 minutes on ice, pelleted, and washed in 250 mM sucrose, 1 mM EGTA, 10 mM Tris-HCl, pH 7.4. The bovine and rat mitochondria were incubated ± 0.5 mM diamide for 5 minutes at 37ºC and then with 50 mM NEM for a further 5 minutes. Mitochondria were pelleted and complex I was isolated by BN-PAGE and non-reducing SDS-PAGE and electrotransferred onto nitrocellulose. [35GSH]- containing protein bands were detected using a phosphor imager. The membrane was blocked in PBST (PBS, 0.05% (v/v) Tween 20 with 1% (w/v) skimmed milk powder) and incubated with ab31832 at 1.25 µg/ml, for 1–2 hours at room temperature. Blots were incubated with 1/5000–1/10,000 dilutions of the appropriate secondary anti-serum for 1 hour at room temperature, treated with the chemiluminescent reagent (ECL or ECL Plus) and vi