Westar eta c 2.0 chemiluminescent substrate

Immunoblotting was performed essentially as described previously (45 (link)). Briefly, proteins were separated by SDS-PAGE on 12% BisTris gels (Invitrogen), transferred to polyvinylidene difluoride membranes (Invitrogen), and incubated with anti-His₆ (Bethyl Laboratories, Inc.), anti-β-lactamase (Abcam), or anti-GroEL (Sigma) primary antibodies followed by an appropriate horseradish peroxidase-conjugated secondary antibody (Sigma). Chemiluminescence was detected using the WESTAR ETA C 2.0 chemiluminescent substrate (Cyanagen) on an Amersham Biosciences Imager 600 (GE Healthcare).

For SDS–PAGE analysis of purified cytb₆f, protein samples were mixed with an equal volume of 2× Laemmli sample buffer (Merck) and boiled for 10 min prior to separation on precast NuPAGE 12% Bis-Tris gels (Invitrogen). For BN-PAGE analysis, cytb₆f was diluted in 4× sample buffer (100 mM Tris–HCl pH 7.5, 0.05% (w/v) bromphenol blue, 40% (w/v) glycerol) and analyzed on precast NativePAGE 3–12% Bis-Tris gels (Invitrogen). Gels were stained with Coomassie Brilliant Blue and imaged using an Amersham 600 imager (GE Healthcare). Alternatively, SDS–PAGE separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (ThermoFisher Scientific) as described previously [37 (link)] and cytochrome c-mediated chemiluminescence was detected using the WESTAR ETA C 2.0 chemiluminescent substrate (Cyanagen) and an Amersham Imager 600 (GE Healthcare).