As the relatively young field of Proteomics continues to develop, it is becoming excruciatingly clear that tackling the entire collection of expressed proteins in any given system is simply too complex a proposition to carry out in one step. One answer to this predicament is to break the proteome up into subgroups, with each subgroup comprised of a more manageable collection of proteins. Depending on the specific goals of the study, any number of fractionation methods are available, with each method relying on some physicochemical property of proteins to effect the fractionation. Common properties of proteins that are exploited in fractionation schemes include size, hydrophobicity, the presence of post-translational modifications (e.g. phosphorylation), and isoelectric point. The current review focuses on fractionation by isoelectric point, or isoelectric focusing (IEF).
While each fractionation method has its own set of advantages and disadvantages, one clear plus for IEF is that the separation is completely independent of the size of the protein, ensuring that the proteins in each fraction are not crowded into one size category. One of the newer devices for solution-phase (or preparative) IEF is the ZOOM® IEF Fractionator from Invitrogen. The fractionator is based on a design reported by Zuo et.al. {Zuo, 2000 #500; Zuo, 2001 #560} and consists of a hollow Teflon tube that is divided into 7 compartments separated by polyacrylamide discs in fritted polyethylene supports. Each of the discs is buffered to a different pH and serves as a solid, yet permeable, barrier to each focused fraction. Discs are available with pH values of 3.0, 4.6, 5.4, 6.2, 7.0, and 10.0. This arrangement yields 5 fractions, each fraction being bordered by an adjacent pair of discs. On each end of the fractionator is a chamber for anode and cathode buffers. Protein sample, in some sort of denaturing buffer with ampholytes, is loaded into the fractionator and voltage is applied. The recommended ramping scheme is 100V for 20 min, 200V for 80 min, and 600V for 80 min. After the run, fractions are retrieved through small ports in the top of the fractionator.
Overall, the device does exactly what it is supposed to do. Complex protein mixtures are separated into 5 discrete fractions. We tested the fractionator on serum and found albumin in one fraction only; a big improvement over solution phase IEF methods that have no physical barriers between the fraction chambers. While the literature recommends fractionating 1-2 mg total protein, we were interested in fractionating tissue lysates of fairly precious clinical specimens. The fractionator performed very well with 250 µg total protein, although we found it necessary to concentrate the proteins in the fractions prior to downstream analysis.
For price, the ZOOM® IEF Fractionator costs about half of what another commonly used solution phase IEF system, the Rotofor (BioRad), goes for. However, the ZOOM® discs are not cheap. Six are required for each run and they cost about $8 apiece. The upside is that the ZOOM® IEF Fractionator is much simpler to run than the Rotofor and requires no cooling. An additional difference is that the ZOOM was designed for fractionation of proteins in a denaturing buffer compatible with downstream 2D-GE, whereas the Rotofor was designed for the fractionation of proteins in their native state. The decision of which device to purchase will ultimately depend on the anticipated sample size (the Rotofor can handle hundreds of mgs) and whether denatured or native proteins are desired.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center