Concentrating Large Volumes With Centricon^® Plus-70 Centrifugal Filter Device

Large-volume centrifugal filter devices are a convenient alternative to stirred cell ultrafiltration devices for purifying proteins from large volumes of solution. Stirred cells are gentle, efficient, and feature a removable membrane for greatest flexibility. However, centrifugal ultrafilters have their own advantages:

• Devices are pre-assembled and ready to use
• Can process 15 – 65 mL volumes; use multiple devices for larger volumes
• Spin times are fast (minutes)
• No need to refill the reservoir (less contamination risk)

The following protocol is an example that demonstrates the use of Centricon^® Plus-70 centrifugal filter devices to purify and concentrate a fusion protein composed of the alpha and gamma subunits of the human high affinity IgE receptor. The alpha-gamma fusion protein is purified and then coupled to Sepharose^® (GE) beads to produce an affinity column for isolating human IgE antibodies. (Note: protocol courtesy of GKT School of Biomedical Sciences, London.)

Method

1. The alpha and gamma subunit components of the high affinity IgE receptor were transfected into mouse myeloma cell line NS0 and the secreted fusion protein was purified on a Protein G column.
2. The eluted proteins were concentrated from volumes of 500–1000 mL to 5–10 mL using a Centricon^® Plus-70 centrifugal filter device in a swinging bucket rotor at 3000 x g for 10 minutes (60 mL per filter unit).
3. Buffer exchange was also performed using the same units in tissue culture grade sodium azide-free PBS.
4. The concentrated samples were filter-sterilized under sterile conditions using a 0.2 μm pore filter.

Results

According to spectrophotometric analysis, 18.8 mg/L of alpha-gamma fusion protein was recovered using the Centricon^® Plus-70 devices (data not shown). The HPLC trace analysis revealed the profile of the alpha gamma fusion component of the human Fc_εRI (See figure below).

HPLC profile of alpha-gamma fusion component of human FcɛRI