HIC elution profile from the 60% ammonium sulphate pellet isolated from S. oleracea , and the SDS-PAGE gel and western blot showing protein content from the elution steps of the HIC purification. (A) HIC elution profile generated by loading the 60% (NH4)2SO4 pellet isolated from S. oleracea; The ammonium sulphate pellet (60% pell; see Figure 1) was loaded onto a 5 mL HiTrap HIC column, washed and eluted as described in Methods. The gradient used for the separation (red line) and the positions of the major absorption peaks obtained by measuring OD at 280 nm (blue line) following the HIC elution profile is shown. Elutions were performed at 500 mM, 400 mM, 300 mM, 200 mM, 0 M ammonium sulphate. (B) SDS-PAGE gel showing protein content from the different steps involved in the HIC purification protocol of Rubisco isolated from S. oleracea; Samples were separated by SDS-PAGE and subsequently stained for protein. The position of Rubisco’s large (LS) and small (SS) subunits are indicated in the figure. 0.6 μg of sample was added per lane. The purity of the Rubisco fractions were calculated as 65.2 ± 4.7% (500 mM), 91.9 ± 3.1%, (400 mM), 92.6 ± 2.2%, (300 mM), 90.2 ± 2.4% (200 mM) and 76.6 ± 3.1% (0 M). (C) Western blot showing Rubisco content from HIC purified S. oleracea Rubisco fractions; The relative intensities of the LS for the four high purity Rubisco fractions were 0.97 ± 0.03, (400 mM), 0.98 ± 0.02, (300 mM), 0.94 ± 0.04 (200 mM) and 0.84 ± 0.06 (0 M). The gels and western blot figures are representative of 3 sample sets.