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).