If you work with purified or partially purified proteins then at one time
or another you will need to perform desalting or buffer exchange, processes that are somewhat similar. Ammonium sulfate, for instance, is used in molar quantities to stabilize and store certain commercially available proteins and also to fractionate native proteins. High salt concentrations, however, are not compatible with downstream applications of pure proteins. Further, in protein purification protocols, the buffer from one step may not be suitable for a subsequent step or application and buffer exchange is
necessary.
There are a variety of methods that can be employed for either desalting or buffer exchange. I have previously used dialysis and ultra-filtration extensively. Dialysis relies upon semi-permeable membranes with defined pore sizes and the equilibration of buffer across the membrane. Ultra-filtration devices also rely on semi-permeable membranes, but work by repeated concentration and dilution of the protein sample. Both techniques can work well and both have advantages and disadvantages.
An obvious alternative for both of these protocols is size exclusion chromatography. Size exclusion works by passing a protein solution over a resin composed of beads that contain small pores. Large molecules, such as proteins, pass straight over the resin while smaller molecules, such as salts, get caught in the pores. There are already several ‘pre-packaged’ products on the market for size exclusion and I have used some of these, however, I have never really been happy with their overall performance. To compete with existing products, Pierce has recently introduced Zeba desalt spin columns.
Zeba columns are pre-packed with a proprietary resin that has excellent protein recovery and desalting characteristics. The exclusion limit is 7 kDa (larger molecules will pass over the resin), hence the column is compatible with most proteins. To drive either desalting or buffer exchange, Zeba columns use a centrifugal format as opposed to either gravity flow or a chromatographic system. Further, Zeba columns come in a variety of sizes and can accommodate protein samples from 2 ul to 4 ml (on 75 ul to 10 ml bed volumes). As a result, Zeba columns are fast to use, extremely efficient in terms of desalting/yield and can be applied to both analytical and preparative scales. Indeed, Pierce claims up to 97 % yield of protein and >95 % retention of salts and small solutes.
In practice, Zeba columns are extremely easy to use; a plug is snapped off the column to allow flow followed by centrifugation in a 1.5 ml, 15 ml or 50 ml tube depending upon sample size. For straight desalting, the sample is applied to the column after the storage buffer is removed by centrifugation, while for buffer exchange, the column is equilibrated with the new buffer before the protein is loaded. As such, desalting can be achieved with two spins in about 5 minutes and buffer exchange requires about 10 minutes and 5 spins.
I have certainly been impressed with the Zeba columns based upon two stand out features of the product. First, the centrifugal format, in conjunction with the proprietary resin, results in excellent yield with no sample dilution. Second, the large variety of column sizes and range of sample volumes make this system very flexible in terms of sample processing. In comparison to available pre-packed, size-exclusion columns, centrifugal columns were all limited to small volumes. In contrast, with large scale, gravity fed columns a low yield of diluted protein was obtained and the process required constant attention. As such, Zeba combines all the best attributes of pre-existing size exclusion columns in one product. Pierce has definitely succeeded in generating an excellent new pre-packed column for size exclusion.
Peter Haggie, Ph.D.
Post-Doctoral Fellow
University of California, San Francisco