The Protean® IEF Cell is a convenient apparatus for quick separation of proteins based on isoelectric point (pI). As compared to the conventional methods of separation, which take approximately 20 hours, Bio-Rad’s Protean® IEF Cell can complete the run in 5 hours, which accelerates research. It is programmable and gives excellent seperation of proteins with consistent reproducibility.
The Protean® IEF Cell is able to run 24 strips of 7cm size or 12 strips of 17cm, which is instrumental in high throughput separation of proteins. It has a maximum voltage of 10,000 volts for faster results. One considerable asset is the ability to control the time/volt hour in each step, current limit, and voltage ramping profile. The system contains 3 preprogrammed methods for new users and can store up to 10 user-defined methods. The peltier cooling system can maintain temperatures from 10 to 250C, which facilitates working with a wide range of proteins. It also offers optional thermal printer records to document the progress of the run. This becomes extremely important in case of a power outage. The printout can also assist in documenting any fluctuations during the run.
The Protean® IEF Cell consists of a focusing tray (7 and 17cm) with a platinum electrode. All the lanes are numbered, which helps in proper identification of samples. A rehydration tray (7 and 17cm) is also supplied; it is used for rehydrating strips overnight and for storing the rehydrated IPG strips. The rehydration tray is also instrumental in equilibrating the strips with buffers prior to 2nd dimension SDS PAGE. The tray is called “disposable” however, we have been using the same rehydration tray for more than 2 years without any problems. Two pairs of forceps, to hold the thin cover on IPG strips, are also included, as are brushes for cleaning the trays after usage.
pH gradient strips (of a variety of gradients) which are compatible with the IEF apparatus are available from Bio-Rad. Although some of these strips are available from other companies, we choose to purchase the strips from Bio-Rad due to the known compatibility with the Protean® IEF Cell, minimal optimization and the thorough notes for usage. The strips are supplied with 12 strips per pack and are competitively priced. The 7cm strips are compatible with Protean® IEF Cell, Mini-PROTEAN® 3 apparatus and Mini-Protean® Dodeca Cell. Bio-Rad also has ampholytes for aiding 2D gel electrophoresis. For pI calibration, Bio-Rad has a mixture of 9 natural proteins, which we use with outdated strips for finding out the drift in pI (if any).
Complete 2D gel electrophoresis can be performed in one day if active rehydration is done. However, our lab uses passive rehydration of strips hence we do 2D in 2 days. We take extreme care in loading the sample, as the sample often comes off the top of IPG strip. This affects the relative amount of protein loaded as the entire sample is not absorbed by the strip. To overcome this we pipet the sample on the top and load it again from the slanting side of the rehydration tray which has helped to minimize loading errors.
We use 1- and 2-dimensional electrophoresis for separation of cytokeratins. Cytokeratins are epithelia-specific intermediate filament proteins. We are studying the qualitative and quantitative differences between cytokeratins expressed in normal and tumor cells. We use proteins isolated from rat tissue and human cell lines. To prepare our samples for electrophoresis, we use high salt and detergent for removal of soluble proteins from rat tissues. We run whole cell lysates from the cell lines. After extraction, we solubilize the sample in solubilization buffer (8M urea, 2M ThioUrea, 2% CHAPS and 1% DTT) followed by protein estimation by TCA precipitation method.
Because of their close molecular weight (e.g. 40 and 70kd) and the homology between different cytokeratins, 2D gel electrophoresis is the best technique to separate cytokeratins. The isoelectric point of cytokeratins varies from approximately 4 to 8, which causes stacking of acidic keratins on one side of a 5-8 strip. Hence, we need to run 2 strips: 5-8 and 4-7, for separation of both acidic and basic keratins. A strip of 4-8 pH gradient would have been great for complete seperation of cytokeratins on one gel. After seperation of cytokeratins we perform immunoblotting using a set of anti-cytokeratin antibodies for analysis of differential expression in altered tissues.
In summary, the Protean® IEF Cell is a costly but quality-assured system. It can be used for high throughput separation of proteins and in elucidating the proteome of altered cells.
Deepak Kanojia
Senior Research Fellow
Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), TMC
Biochemistry and Cell Biology