N dodecyl β maltoside

Inverted cytoplasmic membranes from wild-type R. capsulatus (MT1131) and its mutants were prepared as described previously (Koch et al., 1998a (link), Pawlik et al., 2010 (link)). For Blue Native (BN) PAGE analyses (Wittig et al., 2006 (link)), ICMs (~ 200 μg of total protein) were incubated at 25°C for 15 minutes with continuous shaking (550 rpm Eppendorf Thermomixer) in the lysis buffer (50 mM NaCl, 50 mM imidazole/HCl, pH 7.0, 5 mM 6-aminohexanoic acid) containing 1% n-dodecyl-β-maltoside (Thermo Scientific Inc.). Solubilized proteins were isolated by a 15 min centrifugation step at 70,000 rpm in a TLA 100.3 rotor. The supernatant was mixed with 6 μL of loading dye (5% Coomassie blue G250 in 0.5 M 6-aminohexanoic acid) and 17 % glycerol, and loaded onto 4–16 % gradient BN-PAGE gels (Life Technologies).
For the second dimension SDS-PAGE, the entire lane of a first dimension BN-PAGE was excised and incubated for 30 min at room temperature in equilibration buffer (50 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 6 M urea, 30% (v/v) glycerol). The lane was then mounted on top of a 5 – 15% gradient SDS-PAGE or a 16% Tris-Tricine gel (Wittig et al., 2006 (link)). After western blotting, the membrane was cut into two pieces and the upper part was decorated with α-Myc antibodies and the lower part with α-CopZ antibodies.