Comparative Proteome Analysis by 2D BN/SDS PAGE

One-dimensional Blue Native PAGE (1D BN PAGE) was performed according to Wittig et al. (2006) (link). Mitochondrial membranes were solubilized by digitonin at a concentration of 5g g^–1 mitochondrial protein (Eubel et al., 2003 (link)). For subsequent SDS PAGE, BN lanes with separated protein complexes were transferred horizontally onto SDS gels. Second-dimension PAGE was carried out as outlined previously (Wittig et al., 2006 (link)). Differential gel electrophoresis (DIGE), which is based on labeling of proteins with CyDyes before 2D BN/SDS PAGE, was carried out according to Peters and Braun (2012) .
Two-dimensional IEF/SDS PAGE was carried out as described by Mihr and Braun (2003) . For the IEF gel dimension, Immobiline DryStrip gels (24cm, non-linear gradient pH 3–11) were used. Focusing took place for 24h at 30 to 8000V using the Ettan IPGphor 3 system (GE Healthcare).
For the second gel dimension, IPG stripes were equilibrated for 15min with DTT (0.4g/40ml) and then 15min with iodoacetamide (1.0g/40ml). SDS PAGE was carried out using the High Performance Electrophoresis (HPE) FlatTop Tower-System (Serva Electrophoresis) using precast Tris-Glycine gels (12.5% polyacrylamide, 24 x 20cm).
Gels were fixed for 2h in 15% (v/v) ethanol, 10% (v/v) acetic acid and stained with Coomassie Brilliant Blue G250 (Neuhoff et al., 1985 , 1990 (link)).
Comparative proteome analyses were based on gel triplicates and data evaluation using the Delta 2D software 4.3 (Decodon, Greifswald, Germany) according to Berth et al. (2007) (link) and Lorenz et al. (2014) (link).