Gel electrophoresis is a common method to separate proteins in polyacrylamide gels from mixtures of proteins or cell and tissue lysates. Specific proteins of interest from the lysate can be then purified from the separated gel bands by electro-elution. This method is often used to purify recombinant proteins from a bacterial lysate or the full-length protein from the degradation products. Electro-elution can also be applied for isolating DNA from separated DNA bands in agarose gels. Bio-Rad developed the Model 422 Electro-Eluter to elute protein or DNA and also to perform dialysis for buffer exchange.
Bio-Rad provides the electro-eluter either in combination with the same buffer tank and lid sold with the Protean 3 system or as a single module with the following accessories: glass tubes, frits, silicone adaptors, grommets and stoppers as well as membrane caps. The accessories and two different sizes of membrane caps (molecular cut off of 12,000-15,000 D (clear membrane) or 3,500 D (green membrane) can also be obtained separately. The electro-eluter holds up to 6 glass tubes with membrane caps attached at the bottom, which connect the upper (cathode) and lower buffer chambers (anode) for current flow. Proteins are eluted from the gel pieces in 400-600 µl buffer and can be collected easily with a pipette from the dialysis membrane of the membrane cap. According to Bio-Rad, 70-100% of the protein is recovered after electro-elution.
I have used the electro-eluter routinely to purify tagged recombinant proteins from E. coli lysates; the proteins were first isolated by chromatography, but still appeared as several bands in SDS gels. While the preparative gels were running, I soaked the membrane caps at 60°C in elution buffer for about one hour. Since I purified some of the proteins several times, I could reuse the membrane caps when stored in 0.05% sodium azide (to avoid bacterial contamination) in the refrigerator. When I assembled the electro-eluter, I pushed the frits into the bottom of the tubes, then moved the tubes through the pre-wetted grommet holes; when I used less than 6 tubes, empty holes were filled with the stoppers. It is essential to wear gloves when assembling the caps in order to avoid touching the membrane (which can cause contamination). Then I filled the silicon adaptors containing the soaked membrane caps (12,000-15,000 D) with elution buffer (the Tris/glycine buffer used also for gel electrophoresis); air bubbles should be avoided. The adaptors are then slid over the frosted end of the glass tubes. Any remaining air-bubbles, which interfere with elution, were expelled from the frits by pulling the adaptors on and off. The tank containing the module (lower chamber) was filled with elution buffer to the top of the silicone adaptors, and placed on a magnetic stirrer with a stirrer bar rotating to avoid air-bubbles sticking on the membranes. The glass tubes were also filled to a quarter with elution buffer.
When gel electrophoresis was finished, I stained the gels with a 1% zinc chloride solution, which precipitates SDS, thus staining the polyacrylamide gel white while leaving the protein bands relatively clear. The expected band for the recombinant protein was cut out and diced into smaller slices, which were placed in 3-6 tubes depending on the band thickness. After the tubes and upper chamber (ca. 100 ml) were filled with elution buffer and checked for leakage, the lid was attached and the elution conditions were set (8-10 mA/glass tube for 4-6 hours depending on the percentage of the gel (10 or 12.5%) and the molecular weight of the protein (22 -70 kD). At the end of the elution, the buffer of the upper chamber was removed, the glass tubes taken out of the electro-eluter and the silicon adapters slipped off the tubes. The eluted proteins were pipetted from the membrane and the membranes and silicon adaptors were washed 2x with 100 µl of elution buffer to completely recover the proteins. I usually stained the gel slices with Coomassie, to determine whether the protein elution was complete.
For easy elution of proteins from polyacrylamide gels, I liked to use the Bio-Rad electro-eluter, since there was never leakage of the protein elution (which sometimes occurs in dialysis tubes) and no dialysis tubes had to be boiled in EDTA. But care has to be taken when assembling the unit in order to avoid air bubbles and drying out or contaminating the membranes. The manual contains a detailed description of the procedure, recipes for suitable elution buffers for DNA and proteins from both denaturating and native gels as well as a trouble-shooting guide.