Chelator recycyling
Batho regeneration was achieved via three sequential steps illustrated in Figure 5: Step I: Removal of Tween-20. Red Tween-20 aggregates, from which hIgG was extracted, were resuspended in 200 µl of 0.25 M NaCl, vortexed for 1 minute , followed by short spin (13K, 2 minutes) . The supernatant was discarded and the process was repeated two more times. Step II: Dissociation of the red [(batho)3:Fe2+] complex. The resulting red crystals (Figure 6, A) were resuspended in 800 µl comprised of MeOH\DDW (1:4 ratio) containing 0.25 M EDTA (pH 7) and heated for 3 minutes at 80 °C. The suspension red color, gradually decreased and the solution became colorless and transparent. Step III: Crystal growth. The colorless transparent medium was left to cool to room temperature (20-30 minutes) and further to 4 °C (20-30 minutes). Colorless plate crystals with a range of sizes (20-150 µm) were observed (Figure 6, B).