The BioRad Protean IEF cell is designed to carry out the first dimension of 2-dimensional gel electrophoresis. In other words, it is used to perform isoelectric focusing of protein-containing samples using immobilized pH-gradient gel strips (ReadyStrip IPG strips). The unit consists of a dedicated, programmable power supply with a Peltier-cooled focusing platform. Two pairs of electrical contacts adjacent to the platform permit as many as a dozen 11- or 17-cm, or 24 7-cm IPG strips to be run at the same time. Focusing is carried out in plastic trays that are removable for cleaning.
BioRad sells IPG strips in a variety of pH ranges, including 2 broad range (pH 3-10 linear or nonlinear), 4 narrow range spanning 3 pH units each, and 4 micro range spanning 1.2 to 2 pH units each. The narrow and micro range IPG strips have overlapping pH ranges for creating composite 2-D gel images. After matching protein spots appearing in the overlapping regions, 2 to 4 gel images can be combined to form virtual 2-D gels with excellent resolution.
BioRad sells the IPG strips dehydrated; storage is at –20°C. The IEF cell/IPG strip system can accommodate both passive or active strip rehydration with simultaneous sample loading. For passive rehydration, strips are placed in contact with the protein sample diluted in rehydration/sample buffer (usually a mixture of urea, +/- thiourea, CHAPS or other nonionic or zwitterionic detergent, a reducing agent such as DTT or tributylphosphine, and ampholytes). The strips are covered with mineral to prevent evaporation and left to sit overnight on the bench. Active rehydration, reported to facilitate the entry of large proteins into the gel, is carried out in a similar fashion except 50 V is applied throughout the rehydration period (11 h minimum). BioRad has also recently added cup-loading as an additional sample application method. This is reported to be beneficial when using basic IPG strips, when the samples are contaminated with nucleic acids or other large non-proteinaceous molecules, or when running serum that has not been depleted of albumin. We have used both passive and active rehydration with good results. We have not yet tried cup loading.
Overall, the system is very easy to use, is quite flexible, and performs with excellent reproducibility. The combination of the IEF Cell/IPG strip system with BioRad’s Criterion gels makes high quality 2D gel electrophoresis doable by virtually any lab. At a price of around $7,000, the IEF cell is not cheap, but it does what it is supposed to do. One minor downside that we have experienced is that the attachment of the platinum electrode wire to the focusing chamber is not as robust as it could be. We’ve already broken one and have had to tighten the plastic screws holding the wires to the chamber on more than one occasion. Over tightening the screws will cause them to snap off. We have also found that making our own rehydration/sample buffer and equilibration buffers gives us more flexibility in sample preparation and is more economical than using BioRad’s premade buffers.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center