Step I: Preparation of conjugated micelles
Conjugated detergent micelles were obtained by mixing equal volumes ofmedium A and B as follows: medium A was
prepared by the addition of 6 μL of the amphiphilic chelator
bathophenanthroline (200 mM in DMSO\HCl) and 4.5 μL of
amino acid monomer (200 mM in DMSO\HCl) with 77 μL of:
0.7 mM Brij-O20 in DDW, with vigorous vortexing (10 seconds). 165 μLof medium B , containing 1 mM FeCl2 in 20 mM
NaCl, was then added to medium A with vigorous vortexing and
further incubated for 5 minutes at 25°C. This was followed by the
addition of 13 μL of 1M NaCl and incubation for 5 minutes at 25°C.
Centrifugation (21,000 x g , 5 minutes at 19 °C) was applied, and
the supernatant was removed from the resulting red pellet. The red
pellet was washed once with 30 μL of cold 20 mM NaCl and centrifugation
was repeated (21,000 x g , 5 minutes at 19 °C).
Step II: IgG capture: Freshly prepared conjugated micelles were
resuspended in 100 μL of serum-free medium (Ex-CELL 610-HSF) containing
4% PEG-6000 and the target hIgG at concentration 5 mg/ml. The
suspension was vigorously vortexed for 45 sec and after 10 minutes of
incubation at room temperature, centrifugation (21,000 x g , 5
minutes, 19°C) was applied. The supernatant was discarded and pellets
were briefly washed with 30 μL of cold 20mM NaCl. An additional
centrifugation step followed (21,000 x g , 5 minutes,
19oC) and the supernatant was removed.
Step III: IgG extraction: Conjugated detergent micellescontaining the target IgG were resuspended and incubated with 200 μL of
50 mM Gly, 30 mM NaCl at pH 4 or 200 mM Tris at pH 6.3 in 50 mM NaCl for
10 minutes at 25°C. Centrifugation followed (21,000 x g , 5
minutes, at 19 °C); the supernatant was removed and analyzed by
SDS-PAGE.