Isolation of Chromera Light-Harvesting Complex

C. velia cells were broken and solubilized as described in Kaňa et al. (2016) (link) and then loaded on a fresh, continuous 5–15% sucrose density gradient prepared using a home-made gradient maker in buffer containing 25 mM HEPES pH 7.8 and 0.04% n-dodecyl β-D-maltoside (β-DM). The ultracentrifugation was performed at 140 000 g at 4 °C for 20 h (with rotor SW28, for 40 ml tubes, of an L8-M ultracentrifuge; Beckmann, USA). The resulting band no. 2 contained a strong double band at 18 and 19 kDa, previously identified as ‘fucoxanthin chlorophyll a/c binding protein (FCP)-like antenna’ (Tichy et al., 2013 (link)). The band analysis by Pan et al., (2012) (link) and Tichy et al. (2013) (link) placed this antenna protein within the main FCP-like group of light-harvesting complexes and so it was named Chromera light harvesting complex (CLH).
After separation by sucrose gradient, the antenna protein was desalted using a PD10 column (GE Healthcare) in a buffer containing 20 mM HEPES (pH 7.6) and 0.01% (w/v) β-DM. Spinach LHCIIb was isolated as previously described (Ruban et al., 1994b (link)) and then purified, desalted and eluted in the same buffer as CLH. In both cases, antennas were isolated from samples dark-adapted for 30–45 min.