AKTAprime™ from GE Healthcare (formerly Amersham Biosciences) is a “one box”, compact, automated, liquid chromatography system. The system is easy to install and has very useful cue cards for easy operation. In addition, it has many ready-to-use templates which can be used to create customized methods. The system offers accurate and reproducible results for an array of applications. Among these applications are: desalting, his-tag protein purification, GST-tag protein purification, protein renaturation on a column, purification of monoclonal IgM antibody, purification of monoclonal antibodies using a pH gradient, albumin removal, anion exchange, cation exchange. I have used the anion exchange application extensively for separation of molecules that have negative charge. I performed this in order to purify GST-fusion proteins aurora A, aurora B and anillin. As I used these proteins to make antibodies against them, I needed to obtain proteins with very high purity. Using the AKTAprime™ automated liquid chromatography system, I was able to purify the proteins to a very high purity and as a consequence, the antibodies produced were of the best quality.
The apparatus can be placed in a cold room to keep biomolecules stable; our system is in a sliding door cold cabinet. Fractions are collected in tubes using the fraction collector: a circular rack that is automatically moved from one tube position to the next one. A chart recorder or a computer can be attached in order to trace various parameters such as UV absorbance, theoretical gradient and conductivity, pressure, temperature or pH. The progress of the run and important parameters can be viewed directly on the AKTAprime™ display; parameters can also be printed at any time during a run. With the exception of the gradient, all parameters can also be changed at any time during a manual run.
Using the preloaded templates, one can create fully customized methods for specific applications. All parameters can be changed in accordance with specific applications: time, volume, concentration of buffer, flow rate, fraction base, fraction size, pressure limit, buffer valve position, injection valve position, etc. Customized protocols can be saved for further use. One can store up to 1000 methods. Methods can be also be edited later.
GE Healthcare also offers a variety of columns that can be used for different applications. We purchased a kit with different columns called HiTrap™IEX selection kit containing HiTrap™ DEAEFF column, HiTrap™ CMFF column, HiTrap™ QFF column, HiTrap™ SPFF column, HiTrap™ ANXFF column, HiTrap™ QXL column, and HiTrap™ SPXL column. I found that for purification of GST-anillin the HiTrap™ QXL column worked best.
This apparatus has a very friendly interface and is relatively easy to use once the required parameters, the proper column, settings for chart recorder, and method are set. It allows flow rates up to 50ml/min and pressure up to 1MPA as well as creation of buffer gradients within ample concentrations. However, for optimal gradient mixing, larger chambers must be purchased separately. The sample is injected through the injection valve either with a syringe for smaller volumes or by using a super loop for larger volumes. The sample must be diluted in sample buffer; binding and elution buffers appropriate for the specific application must be prepared in advance.
For faster results, the AKTAprime™ automated liquid chromatography system requires some training from some other member of the lab who has used it before. It requires permanent monitoring as one can lose the sample if elution buffers run out or an unexpected error occurs. I strongly recommend first purifying a small volume of sample and then, if the desired purity is obtained, scaling up to larger sample volumes.
The total duration of a run varies with the protocol used from 37 to 77 minutes plus the time for sample application. For purification of GST proteins, for example, the duration is 37 minutes plus sample application.
To obtain a high purity GST-tagged protein for antibody production, I follow the recommended AKTAprime™ protocol. Briefly, the sample is obtained from classic column purification using glutathione agarose beads and is dialyzed in PBS buffer. I prepare the buffers and check to make sure the system tubing is full of buffer (i.e. no air bubbles). Then I select the appropriate template and parameters. I usually load 1 ml of sample and the run takes about 40 minutes plus 10 minutes to load the sample. I monitor the run about every 15 minutes. It takes about 3-4 runs for me to optimize all the parameters in order to have the desired result. This purification using AKTAprime™ is the last step before sending the protein to have made antibodies against it.
The system requires some maintenance, mainly to make sure that at the end of the run, the system is washed and the loops are soaked in appropriate solutions. There is a preloaded protocol for washing the system between runs called system wash method.
In conclusion, the AKTAprime™ automated liquid chromatography system is a reliable, versatile apparatus that is very efficient for many purification protocols. Using this method, one can obtain a protein with the high level of purity required for various applications.
Dr. Rosalind Silverman
Postdoc
University of Toronto
Department of Medical Genetics and Microbiology