In order to conduct protein analysis of HIV-infected T lymphocytes, our lab used the Bio Rad Protean IEF Cell System in conjunction with the Criterion Cell System to perform 2D gel electrophoresis. Using this system, the entire 2-D gel can be completed within one day (if you are not rehydrating the strips passively overnight). Our Bio Rad Protean IEF Cell is fully programmable, has a 10,000 V power supply with the focusing tray and can run up to twelve 11 cm IPG strips. Some of the custom settings that can be used for each step include temperature control, voltage ramping profile (rapid, linear, slow), time or volt hour control, integral or separate rehydration, programmable current limit, three optional pre-programmed settings, and ten user-defined methods (for up to ten steps). One can utilize passive rehydration overnight or active rehydration (50V) although active rehydration requires the use of the isoelectric focusing tray and cell (passive rehydration can be done in the rehydration/equilibration tray outside the cell).
Conveniently, with the purchase of the Protean IEF Cell, we also received the ReadyPrep 2-D starter kit that included an E. coli protein sample control to help us practice the separation protocols. In addition to the E.coli protein sample, this supplemental kit included rehydration buffer, equilibration buffers I & II, 30% glycerol, iodoacetamide and overlay agarose. We found this kit to be extremely helpful in helping us get our feet wet before using our samples.
We prepared our cell lysate using the Bio Rad rehydration buffer after unsuccessfully attempting to use our own pre-made lysis buffer. Twelve samples of protein lysate were pipetted onto each rehydration/equilibration tray and ReadyStrip IPG strips (in our case, 11 cm pH 4-7) were carefully placed on top of the samples using forceps. This step can be tricky in terms of equal distribution of sample on the strip. We noticed that the samples tended to clump following placement on the tray, an important consideration as samples displaced on top of the strip will not be absorbed. To prevent sample displacement (and thus, ensure absorption), mineral oil was placed on top of the strips. For our experiments, we passively rehydrated the strips with the protein sample overnight (approximately 15 h).
After rehydration, the strips were transported to the PROTEAN IEF focusing tray. It is important to cover the wire electrodes of the tray at both ends of the channel with the wet paper wick (provided) before transferring the strips. The electrode wicks serve as receptacles for salt in the sample and will improve the quality of the IEF step. High salt concentration in the lysate or drying out (incomplete rehydration) may cause problems by burning the strip (or can cause voltage issues). One important safety feature is that the cell only works when the cover is closed. We used one of the three preset methods stored in the PROTEAN IEF Cell to run our sample. In each preset method, the maximum applied voltage is based on the length of the IPG strip and the protocol takes about 51/2 hours for the completion of 1-D.
Following IEF, the strips can be either stored at –70 C or separated using 2-D SDS-PAGE (we used the Bio Rad Ready Gel Precast Gels (8-16%) and the Bio Rad Criterion Cell). Before running the SDS-PAGE, the strips were equilibrated with equlibration buffers I & II in the rehydraton/equilibration trays. The strips were then placed onto the PAGE gel and run for 1 hr at 200 V. Following protein separation, the gels were stained with Bio Rad coomassie and destained with Bio Rad solutions. The Bio Rad PROTEAN IEF Cell was easy to program and follow by viewing the information on the front panel. It was very quiet while running and beeps to indicate a completed run. Another unique feature of the PROTEAN IEF Cell is that one can utilize real time editing to modify a method during a run. One great feature of the Bio Rad Criterion Cell is that the top cover is designed to assist in the separation of the plates following a competed separation by pushing down on it allowing the gel to be easily removed. We obtained beautiful 2-D data and we detected numerous protein spots with minimal streaking. In addition, the Bio Rad technical team was very knowledgeable and provided excellent assistance in helping us to set up our system. It has been a joy using the Bio Rad PROTEAN IEF and Criterion Cells.
There are a few, very minor, disadvantages to this system. As previously mentioned, the equal distribution of sample on the strip proved problematic at times. Second, cleaning the focusing and rehydration trays can be tedious due to the mineral oil, requiring numerous cleanings with detergent (Bio Rad provides the brushes). Third, the accessory reagents are expensive easily costing $400-500 dollars on accessories for a single set of experiments (approximately 12 strips, 12 premade gels, and buffers). Luckily, Bio Rad is very reasonable in providing discounts.
Hee Chul Lee, Ph.D.
NYU School of Medicine