Proteomics-based approaches for the characterization of an entire proteome of an organism require running 2-dimensional polyacrylamide gels. Obtaining well-resolved 2D gels requires that both dimensions be processed accurately and efficiently. The Ettan IPGphor II from GE Healthcare is the ideal option for obtaining perfectly resolved first dimensions of 2D gels. The manufacturer claims that the system provides ‘reproducibility, reliability, speed, accuracy, high protein loading capacity, and high throughput’, and in my experience, the system really stands up to their claim.
To begin with, the system is very compact and can be housed on a tabletop in any corner of the lab. It is perfectly silent and occupies little space. The system requires an ambient, dust-free operating environment; however, we keep the instrument in a well air-conditioned room. The system contains gold-plated copper electrodes that provide the necessary contact to the 1D strips for carrying out isoelectric focusing. A very high voltage is applied between the electrodes and, therefore, caution should be used while handling the instrument. As a safety feature, the electrodes are automatically switched off when anyone opens the lid of the instrument.
We find that setting a run protocol on the instrument is very simple. The system is equipped with a touch button and an LCD screen which is clear and easy to read. We have standardized gradient protocols for different kinds of runs that we store in the instrument. These protocols can be modified as needed. Up to 10 protocols with nine steps each can be set and stored. The run can be monitored at any point during the experiment. The run is generally displayed in V hrs (volts x hrs), i.e. voltage and time are displayed together. The run can be paused at any time and can be resumed from that point at a later time.
The instrument also offers the special feature of being controlled from a computer. The IPGphor II can be connected to a computer via a serial port. The software provided with the system can be installed and used for programming the run. The software offers all the features that can also be directly accessed through the instrument. In addition, the software also makes it possible to control several units through a single computer. However, we do not use the software in our laboratory, since we have only one unit and therefore, we find that a computer interface is not required.
The IPGphor II allows for the simultaneous use of up to 12 strips in a single run; all these strips will be subjected to single program. The maximum length of each strip can only be 24 cm. We have found the ability to simultaneously run several strips useful for processing IEF strips that have been subjected to four different treatments – so, when we run them in triplicate, we can have 12 strips that are resolved using identical electrophoresis conditions. This rules out artifacts of protein separation and gives us good, comparable results. Rehydration of the IEF strip can be done on the IPGphor platform itself. We usually add this step as a part of the run program and this saves us time and also helps to avoid artifacts.
Maintenance of the instrument should be done with care; the system should be thoroughly cleaned up after each use. A detailed protocol for cleaning of the instrument is provided in the manual. The manual also contains complete instructions for running the instrument, including standard protocols for beginners. However, I would suggest that optimization based on one’s experimental needs be carried out to obtain good results. Overall, we find this instrument very good, easy to use and highly cost effective. GE Healthcare has designed the instrument to be very user-friendly even for the proteomics beginner.