An Inexpensive Medium-Scale Preparative Electrophoresis Unit Polyacrylamide gel electrophoresis is one of the most sensitive methods available for the separation of proteins. The gel is subjected to an electric current and the proteins are separated on the basis of their charge to maas ratios as they migrate a t differing speeds through the gel. Under favorable conditions, separation of proteins differing by as little as one amino acid can heeffected. This technique has been used for some years as an analytical tool and has proved so useful that several versions designed for preparative work Y ~ p1.0~ ltube have appeared on the market. Since these units must be capable of accepting large starting volumes, they incorporate many design features that make them relatively expensive (over. $1,000 per unit) and complicated to operate. Some units also require large amounts of ,,,,, electrolyte buffer, which must be made up fresh for each run. A small polyacrylamide gel eleetroohoresis unit was described oreviouslv by Racusen and Calvanicol and advances in rnnt;rials and methds now encburage inhouse cmstruction of such a device when eronumir farttm are rcmsidpred. 'This paper preaenta a low-cost, wmperature-uuntrolled unit which rr easy to budd and operate and providm hrgh resolution iorsmall to medium scale experiments. The unit is made of three parts: upper and lower water-jacketed gel tubes, and a collection ehamher which fits between the gel tubes (see figure). The inner tubes of the upper and lower sections are glass of 2 cm diameter and the lengths are 8 and 4 em, respectively. These dimensions can he varied when the unit is built, of course, depending on the specific application. The water jackets are cut from Plexiglas tubing and the top of the water jacket is sealed with a Plexiglas washer cut to fit snugly between the glass and Plexiglas tuhes. At the other end, the glass center tubes and the Plexiglas water jackets fit into grooves lathe-cut in %-in.thick Plexiglas rings. The center hole of the rings is sized to match the inner diameter of the glass tube. The Plexiglas and glass parts of the upper and lower sections are assembled with epoxy glue. The collection chamber that fits between the Plexiglas flanges is a ring of %=-in.thick Plexiglas, whose center hole matches the inner diameter of the glass tubes. Small holes are drilled laterally from the outer edgeof thedisk to the sides of the inner hole; these are fitted with syringe needles and serve as elution ports. Operation of this unit is much simpler than most commercially available systems. The gel is poured into the upper and lower chambers after the faces of the flanges have been coated with stopcock grease and the gel chambers placed upright on a sheet of glass. After pouring, a thin Layer of water is placed above the gel to eliminate a meniscus. The electrophoresis unit is assembled by coating the flange faces with stopcock grease, placing a piece of membrane cut from dialysis tubing over the lower gel chamber, and bolting the three sections of the unit together through the flanges. The bottom of the lower gel column is placed in a beaker of buffer and a large funnel conneded to the upper glass tube by a short section of Teflon hose serves as the upper buffer reservoir. The amount of buffer in each of the reservoirs can he as low as 500 ml. Sample is layered on top af the upper gel under the huffer via a syringe and plastic tubing. Concentrated protein solutions will sit an top of the gel as a separate, denser phase from the huffer solution. Less concentratedsolutions can be made dense with addition of sucrose. Polyethylene tubing is connected to the elution ports and a current of 400 V is applied across the gel. Throughout the ensuing electrophoresis run, cold tap water is circulated through the upper and lower water jackets to minimize heat denaturation of proteins in the gel. Buffer pumped through the collection ehamher a t 0.6 mllmin sweeps separated proteins moving out of the upper gel column into tubes of a fraction collector. The lower gel column serves to complete the circuit to the lower buffer chamber. Protein entrance into this column is prevented by the dialysis membrane. This unit was used , ~ its simple design and variable dimensions should suit to separate isozymes of frog lysozyme loaded in 1ml ~ a m p l e sbut it equally well to experiments with many different protein and sample sizes. It is a pleasure to acknowledge the generous support provided (J.H.H.) by an NSF-URP grant.
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Racuaen. D. and Calvanim, N.. A n d Rioehem. 7,62 (1964). Snyder. J. A . and Harrimn. J. H., subrnittpd to J. Biol Chern.
Furman University Greenville. South Carolina 29613
J. H. Harrison J. A. Snyder W. C. Harris
Volume 54, Number 8, August 1977 / 487
