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:
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.)
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 FcεRI (See figure below).
HPLC profile of alpha-gamma fusion component of human FcɛRI
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