The process of protein extraction includes using a number of buffers. Ultimately, ultrapure proteins are needed because salt contamination can affect the kinetic studies which often follow purification. Contamination also affects gel electrophoresis; in particular, the presence of the salts has a negative impact on 2D gels. The Centricon filters can also be used as a final step in protein purification, to enhance protein concentration and remove unwanted buffer.
The Centricon Plus-20 Centrifuge Filter Units from Millipore are single use columns intended for quick processing of aqueous biological samples in a volume of 19 ml. The filter unit can be used with a swinging-bucket centrifuge capable of accommodating 50 ml centrifuge tubes. The Centricon Plus-20 can concentrate most 19 ml solutions down to 150 µl in just 15 to 30 minutes.
The Centricon membrane contains trace amount of glycerol (used as humectant). To remove the glycerol, spin 19 ml of sterile of Milli-Q water through the filter. After removal of the glycerol, we place the solution for purification into sample filter cup then close it with the supplied cap. We then set the sample filter cup into the filtrate collection tube followed by the Centricon Plus-20. The entire assembly is then placed in the centrifuge container.
We have used these filters for the purpose of removing unwanted slate and salts from protein samples extracted from bacteria and yeast, as well as from recombinant protein samples. The filters can also be used to concentrate samples. To reduce the volume from 10-15 ml to 200-300 µl, we spun at 4000 x g. Complete concentration may take 15-25 minutes; we monitored every 5 minutes. Viscous solutions may take up to 5 times longer than very dilute solutions. These filters have a wide recovery range; samples with higher protein concentrations may be subject to larger losses.
We found the Centricon filters from Millipore a very useful device for purification of protein samples in a short time. If you are looking for a simple method for desalting protein samples, we suggested Centricon Centrifuge Filter Units.