Protein purification, separation of native conformations of a protein (monomers, dimers, oligomers), enrichment of proteins (e.g. phosphoproteins), etc., are some of the most demanding protocols performed by biochemists in both academia and industry. These are some of the most important and necessary steps for a molecule (protein, receptor) to be characterized or to evolve into the market as a drug target. One of the most convenient and fast ways to purify or characterize a protein is the employment of chromatographic procedures specific to the nature of the protein. Bio-Rad-s DuoFLow Chromatographic system is a boon to scientists for these kinds of applications.
The DuoFLow system consists of 2 peristaltic pumps which can be used at very high pressure for fast resolution of the samples. The pressure can go up to 3500 psi (23 MPa) with a flow rate of 10 ml/min. The user definitely needs to check the chromatographic column specifications to determine the flow rate as well as the minimum and maximum pressure limit allowable under the specific experimental conditions. The instrument comes with a conductivity meter as well as a UV detector along with a fraction collector, all controlled by a common software platform. The fraction collector has an option for collecting fractions at the user’s convenience in different formats, e.g., test tubes, eppendorf tubes. Both the pumps can be used simultaneously or sequentially in a user-defined program mode for optimal efficiency.
There is a sample injection valve which can load samples as little 100 ul, or up to 5 ml using the injection loop. However, large sample volumes can be directly loaded using the pumps also. The samples must be clarified before loading onto the column to maintain the column in its proper condition and this step helps prevent the column from clogging up. After each run, the pumps must be flushed properly so as to keep them clean and in efficient running condition. Care must be taken in order not to introduce air bubbles into the system. Strong methanol washes often help in getting rid of the air bubbles. Another practical tip is to switch off the UV bulb upon completion of the experiment to enhance the bulb shelf life. Also, shutting down the program as well as the computer governing the software helps an easy start during the next run. If the DuoFLow system is stored at 4°C, the instrument should always be left on so as to allow minimum current to flow in order to maintain the circuit and mother board intact.
I have used this instrument linked to a size exclusion chromatography (SEC) column, protein purification columns (His-tag, GST-tag) as well with phospho columns. Programming and setting up the instrument is fairly easy and follows a stepwise logical sequence. Care must be taken to ensure compatibility with the column and its recommended specifications and the protocol in which it is used. The instrument is robust and gives very nice data and resolution. My SEC experiments were run perfectly with this FPLC system and I get wonderful resolution between native conformations of a large size protein which existed in different oligomeric states. Protein purification experiments were also a good success.
Communication between the instrument, software and the fraction collector must be ensured before each run as errors tend to pop up. This is one aspect which I found troublesome at times and had to restart everything. The good part of the instrument is that it resolves and translates the data very quickly. Although expensive, it is a good buy for any standard lab working a lot on protein biochemistry. Many accessories come with the basic instrument which can be supplemented depending upon the demands of the experimental set up.