Small-scale Antibody Purification with Buffer Exchange and Concentration: Amicon^® Pro System

If an antibody must be purified from a small sample volume (0.5-10 mL), then the Amicon^® Pro Purification System is recommended. Because this centrifugal device enables purification, buffer exchange, and concentration to be performed without transferring the sample or diluting it, the risk of antibody loss is minimized. The following method was developed for affinity purification of antibodies (0.5 mL sample) using protein A/G agarose resins in the Amicon^® Pro System.

Method

1. Add 200 µL Protein A/G Resin (50% slurry) and 1.5 mL Wash buffer to exchange reservoir. Spin 1000 x g for 1 min.
2. Add 0.5 mL sample and mix with resin by pipetting. Incubate with upright agitation in an orbital shaker for 1 h. [Note: end-over-end mixing may be less effective, because residual resin on the wall and cap may not be collected efficiently.]
3. Spin 1000 x g for 1 min to clear unbound species.
4. Add 1.5 mL Wash buffer. Spin 1000 x g for 1 min.
5. Attach the Amicon^® Ultra 0.5 mL filter (10k MWCO).
6. Add 1 mL Elution Buffer and mix with resin. Spin 4000 x g for 10 min.
7. Add 1.5 mL final buffer of your choice. Spin 4000 x g for 15 min to exchange buffer and concentrate.
8. Remove Amicon^® Ultra filter and place into filtrate collection tube.
9. Recover purified antibody from the Amicon^® Ultra 0.5 mL filter by reverse spin.

Results

IgGs from rabbit serum (Cat. No. S20-100ML) were purified on Protein A or Protein G agarose, using either the Amicon^® Pro System (10kDa MWCO, Cat Nos. ACK5010PA and ACK5010PG) or traditional spin columns. Purified IgGs were then buffer-exchanged and concentrated, either in the same Amicon^® Pro devices or transferred from spin columns to separate Amicon^® Ultra devices.

When the Amicon^® Pro System was used, the process required 6 pipetting steps/spins, as compared to 11 pipetting steps/spins required for traditional spin columns and concentrators. As shown in the figure below, antibody recovery remained unchanged.

Antibody purification from rabbit serum using Amicon^® Pro System and traditional spin columns (n=3)