Pierce’s iCON concentrators are easy to use and fast. They can be used for a number of purposes. Samples can be concentrated into smaller volumes, buffer exchange can be performed, as well as desalting, and the removal of unincorporated reagents that are used to label proteins can be accomplished. The concentrators contain a “ high-performance regenerated cellulose membrane” that the manufacturer claims results in a high surface area that minimizes protein polarization and adsorption. These membranes are contained in either a 15 or 50 ml tube with a 7 and 20 ml volume capacity, respectively; molecular weight cut-offs are either 9K or 20K Daltons. My experience is with the 20K unit and 20 ml volume capacity employing tissue culture media containing monoclonal antibody or purified monoclonal antibody preps for the purpose of removing low molecular weight contaminants and concentrating the antibody or for buffer exchange. Biological samples such as enzymes and DNA can also be used with the concentrators.
The concentrators are used by: adding your sample to the upper sample chamber, centrifuging the tube, and collecting the remaining liquid in the upper sample chamber (retentate). The upper sample chamber contains molecules larger than the molecular weight cut-off (MWCO) and those below the MWCO pass through the filter to the bottom of the tube. The concentrators can be used with either a swinging bucket or fixed angle centrifuge, although centrifugal speeds and volumes used differ between the two.
The concentrators are “single use” devices. One type of sample should be used for each tube, but separate aliquots of this sample can be run through the same concentrator a number of times. This method can be used for buffer exchange/diafiltration by adding a sample, centrifuging, and diluting the sample from the upper sample chamber to its original volume with exchange buffer and centrifuging again, up to 4 or more times. Other than for buffer exchange, I have not found it necessary to centrifuge the sample for concentration more than 2-3 times or for more than 5-15 minutes each time.
In fact, there have been times when my sample became too concentrated and began to precipitate on the filter. Adjusting the dead stop volume, a method recommended for preventing over-concentration of proteins, can prevent this problem. The filters will stop concentrating the sample when the volume in the bottom of the collection tube is at the meniscus of the upper sample chamber. The dead stop volume can be adjusted by the amount of liquid added to the bottom chamber before centrifuging.
Some sample loss does occur when using the iCON, especially when performing buffer exchange, which requires multiple centrifugal spins. The manufacturer claims >90% recovery and a concentration factor of greater than 150-fold in less than 30 minutes. In fact, in a data table on the manufacturer’s website, 91 and 98% recovery of 2 different proteins using the iCON 9K MWCO was shown to be a greater protein recovery than two other competitors.
Time may be a factor as this method is much less time-consuming when processing multiple samples and can be used for volumes up to 20 ml. The iCON concentrators provide a fast and easy alternative to conventional techniques for buffer exchange, concentrating, desalting and removing unincorporated reagents used to label proteins. I found a far superior concentrating factor when compared to other brands of filters.
Research Associate
Cell Culture, Quality Control Department
Theranostech, Inc