The BioRad Rotofor Cell System is designed to perform solution-phase, preparative isoelectric focusing of complex protein mixtures. The Rotofor Cell consists of a cylindrical focusing chamber into which fits a plastic core that divides the chamber into 20 compartments separated by polyester screens. The protein sample is diluted into a mixture of water and ampholytes and loaded into the assembled chamber. Non-ionic detergents may also be included if required to enhance the solubility of specific proteins. At the recommended power of 12-15W, a standard focusing run takes about 4 h. Voltage increases throughout the run until focusing is complete. Monitoring the voltage over time is a convenient method to gauge the progress of the run. Fractions are recovered directly into test tubes using a vacuum manifold.
There area number of advantages of the Rotofor system over other fraction techniques. Perhaps the two most important are the retention of the protein’s biological activity when using the Rotofor system and the ability of the Rotofor to handle large quantities of total protein. For these reasons, it is an ideal first step in protein purification schemes, especially those focusing on low abundance proteins. BioRad claims the successful isoelectric fractionation of 2.4 g lyophilized cell culture supernatant. A caveat, however, is the fact that some proteins precipitate at their isoelectric point. Serum albumin, due to its high concentration in serum and its specific physicochemical properties, forms a large, gummy precipitate on the polyester screens separating the fraction compartments. This deposit must be removed manually (usually by scrubbing with a toothbrush) prior to the next run. Other than this issue, we routinely fractionate 120 mg serum protein with good results and reasonable reproducibility.
Although for the most part the Rotofor system performs what it is designed to do, there are some features to be aware of when using the system. For one, the unit must be assembled very carefully to ensure leak-free seals. Although this is something that becomes routine with practice, novice users may experience leaks or may find that the assembled unit doesn’t fit into the housing. We have also found that, at least with the mini-core, the sample chamber must be filled to the absolute maximum in order to avoid bubbles that can cause the power supply to shut off. We have also found it necessary to be very diligent about replacing the vent buttons after every 5 runs. Although this is recommended in the instructions, it is easy to forget and will lead to leaks due to pressure build-up. Finally, focused protein mixtures exhibit considerable overlap between fractions. Some proteins may appear in 4 or 5 fractions rather than in more defined regions as is possible with IPG strips.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center