Deae sepharose anion exchange column

Cytochrome c oxidase (COX) was purified from porcine heart following our established protocol^{19 (link),73 (link)} which preserves post-translational modifications. Briefly, the porcine heart was homogenized, and the tissue extract was loaded onto a DEAE-Sepharose anion exchange column (GE Healthcare) equilibrated with 125 mM phosphate buffer and 0.5% Triton X-100, pH 7.4. The bound enzyme was eluted from the column using a salt gradient going from 100 mM to 700 mM phosphate buffer with 0.1% Triton X-100, pH 7.4. The enzyme was further purified via ammonium sulfate precipitation.

The fish species barfin plaice (Liposetta pinnifasciata), longsnout poacher (Brachyopsis rostratus), notched-fin eelpout (Zoarces elongatus Kner), and saffron cod (Eleginus gracilis) were collected by Nichirei Corporation. Size exclusion chromatography and anion exchange chromatography were successively performed for the prepared crude sample powders with a Sephadex G-25 size-exclusion column (XK 50/30, 500 mL; GE Healthcare Life Sciences, Pittsburgh, PA, USA) and DEAE Sepharose anion-exchange column (XK 50/20, 80 mL; GE Healthcare), respectively [7 (link)]. The purified samples were dialyzed against Milli-Q water for three overnights, and then lyophilized for frozen storage. The purity of the final product was checked with 15% SDS–PAGE using a minislab electrophoresis kit (AE-6500; ATTO Corp., Tokyo, Japan).