Chromatography is a widely used method for separation of proteins or peptides from mixtures or different cell or tissue lysates. Since chromatography can be a time-consuming method, especially for preparative purification when larger volumes of sample (e.g. antibody purification from supernatant), washing or elution buffer are used, it is convenient to automate the process. For this purpose Bio-Rad developed the Econo Gradient Pump, a 2-channel low-pressure pump, which in combination with different valves can be used for low-pressure chromatography purification, like affinity purification of tagged or non-tagged recombinant proteins, or online bio-assays to measure enzymatic/biological activity with substrates. Proteins can be sampled after isocratic or gradient elution with high accuracy to assure reproducibility.
The Bio-Rad Econo Gradient Combos are assembled from the following: Econo Gradient Pump (available for 100/120 V, cat. # 731-9001) or 220/240 V (cat. # 731-9002), the gradient mixer valve (Combo 1, cat# 731-9030 or 731-9032), a rack with column clamps (Combo 3, cat# 731-9038 or 731-9040) and MV-6 injection valve (Combo 2, cat# 731-9034 or 731-9036). These parts can also be bought separately, depending on the user’s needs and budget. Different accessories can be purchased to extend the system: a SVT-3 diverter/bypass valve (3-way valve) or a splitter valve kit, which includes tubing, adaptors and a 'T' union back-pressure valve, a pump fittings kit, a UV monitor, different fraction collectors, a chart recorder as well as cables for connection to fraction collectors and chart recorders from Bio-Rad or other vendors.
The Econo Gradient Pump can be used with flow rates of 0.002-20 ml/channel. The pump can also be used to control the gradient mixer for binary gradient formation and the diverter valve for waste and fraction collection; you can also use it to hold/pause the collector. The pump can be easily calibrated for different tube sizes and programmed for different chromatography runs including gradient formation.
We bought the Econo Pump with a UV monitor, column rack, injection and diverter valve in order to purify his-tagged recombinant proteins expressed in E.coli with immobilized metal ion affinity chromatography (IMAC). Several proteins needed to be purified from bacterial extracts for further identification as antigens via T cell assays and injection into rats. When we got the Econo Pump and accessories, assembly of the system, connection with our own fraction collector and chart reader, and calibration for the tube size, were easy to perform. This was due to the detailed instructions in the manual.
For purifications, we easily programmed the Econo Pump to load the sample on a nickel-equilibrated chelating sepharose column, wash the column with 20x column volume of urea buffer, pH 8.0 (flow rate 0.4 ml/min) and elute the proteins with a pH-gradient of urea buffer (pH 6.0-3.0). The protein amount in the elution buffer was monitored by the UV detector and plotted by a chart recorder before the elution buffer was collected in 3 ml fractions. Using the Econo system, protein isolation was very accurate, so that the eluted proteins of interest were collected in almost the same fractions in each purification. This allowed us, after a few purifications, to analyze fewer fractions for the presence of our proteins of interest. Sometimes, when the urea buffer started to crystallize and clog the tubes, the fraction collector would stop or the built-up pressure would disconnect the tubes, so that the buffer and sample would spill onto the table. Therefore, I found that checking the tubes for crystallized urea and the connections for a tight fit, or exchanging tubes after several purifications to avoid leakage was important. We used the system continuously on a daily basis, but never experienced any other problems.
Therefore, I recommend the Econo Pump and its accessories for easy and accurate chromatographic purification of proteins, especially since parts can be purchased separately, so cost can be reduced to suit the researcher’s needs. Bio-Rad's website contains information about the pump combos, the accessories and tubes as well as the manuals. When the system arrives, you can call to receive assistance from a Bio-Rad representative in assembling the system.