Millipore Centricon® Centrifugal Filter Units are very useful for concentrating proteins, exchanging buffer, desalting, and removing of impurities. Centricon® filters come in five variations, each with a different molecular weight cut-off (3, 10, 30, 50 and 100 kDa). Smaller and larger volume columns are available. The molecular weight cut off of every column can be identified by its color-coded O-ring. A low binding membrane has been employed in the construction of this column with a patented deadstop technology. This allows minimal loss (up to 70% protein recovery is possible) and prevents complete flow through of the protein sample. Once the volume reaches ~ 50 ul, there is no more flow through even if the centrifugation is carried out for longer time.
I have used these columns for the past 5 years, primarily for concentrating proteins. I have concentrated purified proteins as well as proteins from whole cell plant extracts. These concentrated proteins were used for injecting into mice for studying the generation of neutralizing antibodies. The ability of these columns to concentrate protein was to my utmost satisfaction due to their ease of operation. I only needed a refrigerated centrifuge with a fixed angle rotor. Recently, I have also used these columns for buffer exchange: 2 ml of protein (maximum limit for the columns I used) can be concentrated to 50 ul. Another way these columns can be used is for purification of protein fragments smaller than the molecular weight cut-off. In my hands, however, this application has not been satisfactory. I have seen that protein fragments of lower molecular weight were also concentrated along with the desired protein (i.e. the smaller molecular weight fragments did not separate from the larger protein).
In order to minimize protein loss, I recommend running 2% Bovine Serum Albumin (BSA) through the column before loading the protein sample so that none of the desired protein is lost by non-specific binding to the membrane or column walls. Another recommendation I have is to collect the concentrated sample by performing an inverse spin where the column is inverted and centrifuged at low speed. I collect in this manner as I have some reservations about the cleanliness of the retentate caps, which apparently are not clean and sterile.
Another negative feature is inability to use the 2 ml columns in RC5B centrifuges with SS-34 rotors which are very commonly used in labs; the lid cannot be closed if the retentate cap is put in place.
The overall rating of these filters is good. I will recommend them device over dialysis cassettes and tubing.
Centricon® Centrifugal Filter Units From Millipore
Excellent tools for protein concentration and buffer exchange.
Protein loss but that also occurs with every other technique employed for this purpose.
The Bottom Line
A versatile, fast and reliable tool for protein concentration and buffer exchange, with no mess of buffer tanks and dialysis membranes – just the Centricon® filter and a centrifuge.