A modified preparative gel electrophoresis apparatus with special application to the separation of long polypeptide chains

ANALYTICAL

BIOCHEMISTRY

A Modified

62, 461-471

(1974)

Preparative

with

Gel

Electrophoresis

Apparatus

Special Application to the Separation of Long Polypeptide Chains OTTO VOGEL1

Max-Plan&-Institut Received

AND

fiir January

HORST Biologic,

29, 1973;

REINMUTH Tiibingen,

accepted

July

Germany’ 1, 1974

An apparatus for preparative electrophoresis is described which modifies earlier designs substantially. The apparatus is applicable to both continuous and discontinuous buffer systems. Its efficiency is demonstrated for the separation of the three components of the pyruvate dehydrogenase complex. The modifications are discussed with respect to earlier designs.

Since the original introduction of polyacrylamide gel as a separation medium for biological substances simultaneously by Raymond and Weintraub (1) and Ornstein and Davis (2) numerous attempts have been made to use this method on a preparative scale. A large variety of suggestions, designs, and improvements has been presented, especially concerning elution, cooling, homogeneity of the electric field, and technical simplicity; most of this work has been reviewed recently (3-5). However, a basic limitation of preparative electrophoresis [for definition see (6) ] is that only small amounts of material can be applied per unit gel surface before overloading occurs. Therefore, the preparative apparatus requires an enlarged cross-sectional area which accentuates other problems. Among these the most dominant are heating due to electrical resistance and in designs with continuous elution, inefficient elution. During studies with the pyruvate dehydrogenase complex (PDHC) from Escherichia coli we found that the most convenient procedure for separation of the component proteins was polyacrylamide gel electrophoresis in sodium dodecylsulfate (SDS). Because of the high molecular weight of the component polypeptide chains (between 56,000 and lOO,O~ 1 Present address : Biologisches Institut I der Albert-Ludwigs-Universitit, D-7800 Freiburg i. Br., Katharinenstrasse 20, Federal Republic of Germany. ‘Requests for reprints should be sent to: Horst Reinmuth, Max-Planck-Institut fiir Biologie, Abt. Henning, D-7400 Tiibingen, Corrensstrasse 38, Federal Republic of Germany. Copyright All rights

@ 1974 by Academic Press, of reproduction in any form

461 Inc. reserved.

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daltons) long run times were needed. Using different devices (6-8) we found the principle suggested by Jovin et al. (6) the most efficient one for our purposes. Nevertheless, for long runs this design proved unsatisfactory in several respects (see Discussion). We therefore developed an apparatus based on the same principle (i.e., a gel tube as a matrix with central elution of the separated material). Our design provides the following features: A symmetrical elution system in which the elution buffer enters the elution chamber equally from all points on the periphery and is undisturbed by air bubbles even during long runs. The cooling of the gel is not affected by the elution cavity. Completely stable hydrostatic conditions even for low gel concentrations. This is due partly through thin gel holder pins and partly to the low buffer height in the upper electrode vessel (normally 3 cm). Soft gels are stabilized hydrostatically by the height of the elution buffer in the elution cavity. An efficient electrophoresis buffer circulation connected to a large buffer reservoir. This arrangement provides an almost unlimited buffering capacity. It is applicable to both continuous and multiphasic buffer systems (see operational procedure). Cooling through the electrophoresis buffer circulation system. The cooling efficiency is increased by cooling the upper gel surface. The design implements an essential reduction of the operational steps to start an electrophoresis run and enables quick manipulations during the run. We report here the technical details of the apparatus and demonstrate its efficiency for the separation of the three components of the PDHC from E. coli. MATERIALS

Proteins. The PDHC was used for separation. It was purified from E. coli K-l-l LR-813 (9) as described elsewhere (10). Chemicals. Acrylamide and methylene bisacrylamide came from Eastman Kodak (Rochester, NY) ; N,N,iV,iV-tetra-methylene diamine, ,&mercaptoethanol, sodium dodecylsulfate (“reinst, salzfrei”) from ServaEntwicklungslabor (Heidelberg, Germany) ; Tris (hydroxymethyl) aminomethane (“Trisma base” reagent grade) from Sigma (St. Louis, MO.) ; Coomassie brilliant blue from Schwarz/Mann (Orangeburg, NY) ; all other chemicals (p.a. grade) were from E. Merck AG (Darmstadt, Germany). Technical materials. Perspex (polymethylmethacrylate or Lucite)

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and Acrifix 92 came from Roehm GmbH (Darmstadt, Germany), polyamide from A. Reiff KG (Reutlingen, Germany) ; the Pyrex glassware elements were manufactured by E. Biihler (Tiibingen, Germany) ; the glass membrane (Corning no. 7930 glass) plus the rubber gasket was obtained from Buchler Instruments (Fort Lee, NJ) ; UHU-plus was from H. u. M. Fischer GmbH (Biihl/Baden, Germany). METHODS Analyticnl

Electrophoresis

Essentially the same procedure as that described by Weber and Osborn (11) was used, except. that the gel buffer was 0.05 M sodium phosphate pH 7.2, 0.05% SDS and the electrophoresis buffer was 0.02 M sodium phosphate pH 7.4, 0.02% SDS. 70 X 5 mm glass tubes were used. Runs were made at 5 mA/tube at room temperature. After electrophoresis and staining the gels were destained in a Canalco destainer (Canalco, Rockville, Md.). For analytical analysis of fractions separated by aliquots were mixed with one-tenth of a preparative electrophoresis, solution containing 1% SDS, 1% P-mercaptoethanol, 0.05% bromophenol blue, and 20% glycerol and were applied to the analytical gels. Preparative

Electrophoresis

Description of the apparatus. A vertical cross section drawn to scale is shown in Fig. 1 and a horizontal cross section at the level of the marks in Fig. 1 is shown in Fig. 2. The whole apparatus has a diameter of 15 cm and a height of 23 cm. The cross-sectional area of the gel is about 15.7 cm2, but from our experience to date we believe that the cooling system is efficient enough to tolerate a cross-sectional area up to about 20 cm?. Normally one has to handle only four components: (1) the apparatus cover plate containing the cooling finger with the upper electrode, (2) the cylindrical electrophoresis chamber block, (3) the gel polymerising plate or the glass membrane plate, and (4) the lower electrode vessel with the lower electrode. The cooling finger is exchangeable and firmly fixed by the upper O-ring. The essential innovation in this cooling finger compared with earlier designs is the presence of three holes in the outer glass tube to allow mixing of the coolant and electrophoresis buffer systems (see Operation Procedure). The cylindrical electrophoresis column is surrounded and held by a cooling jacket. Inside the cooling jacket an additional tube is inserted to direct the flow of the cooling buffer to the overflow of the cooling jacket in order to obtain uniform cooling. The cooling jacket is sur-

464

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AND

REINMUTH @ubon

Elutiy;,;tuffer

buffer

outlet

-=

Holes for buffer outlet

COVef Elution eve Cooling Electrode reservoir

jacket

electrode

Elution

captllary

Resolving

-

chamber

gel

lindrical projections (gelholder pins)

buffer (coolant)-m Elution

Upper

chamber/

Lower electrode chamber

Membraie holder

membrane Stirriig

\

Rubber gasket

Buffer upper

outlet for electrode chamber

Basal Lower

ring electrode

1%

bar

FIG. 1. Vertical section diagram through the cent,er of the apparatus; the mat&& used are (m) glass; (m) perspex; (W) polyamide; (m) rubber. Interruption of the perspex hatching meaus that the material is not glued at those points. c level of the horizontal cross section shown in Fig. 2.

rounded by and fixed to the outer part of the electrophoresis chamber block which consists of two pieces, the lower one containing the thread holding either the polymerising plate or glass membrane plate. The space between this part and the cooling jacket is connected directly to the elution chamber and serves as the elution buffer inlet. By adjusting the height of the elution buffer inside this space to the same level as the buffer

Glass

Fra. 2. Horizontal mark in Fig. 1.

section

diagram

through

tube

Cooling

finger

Elution

cavity

the

apparatus

at

the

level

of

the

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in the upper electrode chamber (see below) the hydrostatic equilibrium in the whole gel-buffer system is maintained. The electrophoresis column has three overflows at exactly the same level allowing the electrophoresis buffer to flow from the upper electrode chamber to the cooling jacket and from there to the lower electrode chamber. The polymerising plate or glass membrane plate is screwed into the lower part of the electrophoresis chamber block with a special wrench (Fig. 3a-c). In the case of the polymerising plate, a rubber O-ring is used to tighten up the space for the gel during polymerisation. The position of the eIectrophoresis chamber block inside the lower electrode chamber and the arrangement of the lower electrode follow the suggestion of Gordon et al. (7). During operation the apparatus is placed on a leveled magnetic stirrer. Operational procedure. For polymerisation of the gel, the polymerising plate is inserted into the bottom of the gel chamber. The three holes in the cooling finger are plugged with cork stoppers, the central capillary of the cooling finger is filled with water and the elution buffer outlet tubing is clamped. The cover plate with the cooling finger is then put on

a

b

FIG. 3. Accessories for the apparatus: (a) polymerising holder plate; (c) wrench for insertion of (a) and (b).

plate;

(b)

glass

membrane

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the electrode chamber block and fixed with screws as shown in Fig. 1. The cooling finger is centred by the conical pin in the polymerising plate and its height is adjusted by screwing it through the cover plate. For polymerisation the cooling finger has to be pressed firmly against the polymerising plate ; then it is fixed by the fixation screw (see Fig. 1). The gel chamber may be examined for leaks by filling the elution cavity with water. The gel chamber block is now ready to be inserted into the lower electrode vessel which is placed on the leveled magnetic stirrer. Then the electrophoresis buffer-coolant system may be added. To this end, the electrode buffer inlet (see Fig. 1) is connected to the electrode buffer reservoir for the upper electrode chamber through a peristaltic pump (capacity 1 liter/min). The buffer is conducted from the coolant outlet (see Fig. 1) to the cooling jacket by a polyethylene tube through one of the holes in the cover plate. In the case of a multiphasic buffer system, the buffer flows from the cooling jacket through tubing running from the overflow of the cooling jacket, through the wall of the lower electrode vessel and back to the same reservoir. A separate buffer reservoir is connected to the lower electrode vessel through the same pump which operates the circuit for the upper electrode vessle. In the case of a continuous buffer system, only one buffer reservoir is used. Here the electrophoresis buffer flows from the cooling jacket into the lower electrode vessel and back again into the buffer reservoir. Polymerisation of the gel should be done at the same temperature as the run; in our experiments it always was 4°C. The gel is added through a hole in the cover plate and overlayered with water, and the circulation of the precooled buffer (4°C) is started at 1/z liter/min. After polymerisation (about 1$&--l h), the unpolymerised material is discarded and the gel overlayered with the gel buffer. Before removal of the polymerising plate the elution buffer outlet tubing has to be unclamped to allow pressure equalisation in the elution chamber. The polymerising plate has to be unscrewed very cautiously at first. Damage to the lower gel surface is precluded by a completely smooth, carefully polished surface on the polymerising plate, which is covered with a thin film of Vaseline. The gel is held not only by adhesion to the glass wall but also by mechanical support from perspex holder pins (see Fig. 1). From our experience these gel holder pins are without noticeable effect on the quality of the electrical field. We have never observed any distortion of the bromophenol blue band nor of the hemoglobin band in a SDS-free buffer system. AS the next step, the glass membrane plate is screwed into the electrophoresis chamber block, until the rubber gasket around the glass membrane seals securely to its perspex counterpart in the electrophoresis chamber block (see Fig. 1). In this design the elution chamber has an

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ELECTROPHORESIS

467

invariable height of 3 mm. This we found sufficient for all purposes (for further comments see Discussion). Before the electrophoresis chamber block is inserted once more into the lower electrode chamber, the latter is filled half full with electrophoresis buffer. It is important that the remaining air below the glass membrane is completely removed. This can be achieved by vigorous stirring in the lower electrode chamber. After filling up the upper electrode chamber and the elution system with the corresponding buffers, the sample can be added and the electrophoresis started. At the same time the cooling system, the elution system, and the magnetic stirrer are switched on. Hydrostatic equilibrium across the gel is established by adjusting the tip of the elution buffer outlet tubing to a height which brings the buffer in the elution tube to the level of the buffer in thz upper electrode chamber. When all the components of the sample have migrated into the gel (about M hr) the run is interrupted for a moment. The cork stoppers are removed from the holes in the cooling finger with tweezers. (Earlier removal of the stoppers would mix up the overlayered sample with the electrophoresis buffer, circulating through the upper electrode chamber.) Now the electrophoresis buffer can flow through the apparatus as described above being extensively mixed in the corresponding electrode buffer reservoir (s) . Construction remarks. The perspex parts were glued together with ACRIFIX 92. The electrophoresis column was sealed to the corresponding perspex parts with UHU-plus at 60°C which gave a perfect and permanent bonding (see also legend to Fig. 1). Experimental conditions. The experimental conditions were mainly the same as described elsewhere (10). For the gel buffer a Tris-Cl concentration of 0.1 M instead of 0.37 M was used. A current of 3 mA/cm2 was applied, resulting in a potential gradient of about 20 V/cm. The most convenient procedure for concentrating the combined fractions after elution proved to be lyophilisation to about l/10 of the starting volume followed by extensive dialysis against 0.001% SDS, 0.05 M (NH,) HCO, (for further experimental details see legend to Fig. 4). RESULTS

Separation of the PDHC-components. We found the PDHC to be an appropriate system for testing the performance of the electrophoresis apparatus. The molecular weights of the component polypeptide chains (lOJ2) cover an appropriate size range and the amount of each component present in a fraction can be estimated directly from their staining intensity with Coomassie brilliant blue (13). Figure 4 shows one of our separations. As can be seen from analytical test runs of the separated

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0.6

2i0 ml

Eluate

2i0

2jO 300

360

420

-

FIN. 4. Separation of PDHC by preparative polyaerylamide gel electrophoresis at 4°C. The applied sample contained about 65 mg protein in 5 ml 0.01 M Tris-Cl buffer, pH. 7.5, 0.1% SDS, 0.01% P-mercaptoethanol. The elution rate was about 35 ml/hr, 5 min fractions were collected. The dihydrolipoamide dehydrogenase was eluted after 8 hr, the lipoamide transacetylase after 10 hr, the pyruvate dehydrogenase after 13 hr. The lower part of the diagram summarises the adsorbancy at 280 nm for the combined fractions as indicated. The peak for the lipoamide transacetylase is comparably small corresponding to the low adsorbancy of that protein at 280 nm. For analytical test runs 90 ~1 of each fraction were mixed with 10 ~1 of a solution containing 1% SDS, 1% P-mercaptoethanot, 0.05% bromophenol blue, and 20% glycerol and the mixtures applied to analytical gels. The upper part of the diagram shows the results of the test runs. The single photograph on the left of the diagram is taken from a gel loaded with 30 pg of PDHC as isolated from cells. The bands from top to bottom of the gel represent pyruvate dehydrogenase (MW lo5 daltons), transacetylase (MW 8 X 10’ daltons), and dihydrolipoamide dehydrogenase (MW 5.6 X 10’ daltons), respectively. Some minor contaminations are present. The photographs on the right of the diagram show the protein patterns in the corresponding fractions. Overloading is responsible for some of the overlapping of the separated peaks. The arrow and the hatching indicates the fractions containing the dye marker.

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fractions, very pure (>95$%) fractions from all components were obtained. Between 60 and 70% of every protein was recovered better than 90% pure. These calculations were based upon a technique, described elsewhere (13), which gives absolute protein values of each component of the PDHC. As can be seen from Fig. 4 the three components are eluted after 8, 10, and 13 hr, respectively. DISCUSSION

For a preparative gel electrophoresis apparatus with elution of the separated substances during the run, the principle first proposed by Jovin et al. (61 is the most evident one because it is radially symmetric. This is important particularly for the efficiency of heat dissipation and elution, because there is no longer distance from any point in the cross-sectional area to the cooling finger containing the elution capillary than the radius of the electrophoresis column. Many of the numerous modifications of the original design were undertaken in order to improve the elution efficiency and to facilitate construction and operation. Working with some of those designs we felt that the following problems had to be attacked more specifically: (i) symmetry of elution, (ii) trapping of air bubbles in the elution chamber during the run, (iii) stability of the hydrostatic conditions, (iv) increase of the buffering capacity particularly in respect to long runs, (v) reduction and facilitation of the operational steps. The suggestion of an annular elution tube around the electrophoresis column was already made by Jovin et al. (6), who mentioned its importance for the removal of air bubbles in the elution chamber. But it was abandoned by the commercial model because of the relative inefficiency of cooling (14). Using perspex for most parts of the apparatus, we could solve this problem by moving the elution tube to the outside of the cooling jacket. The efficiency of cooling at the lower part of the electrophoresis column is increased by the perspex tube inside the cooling jacket. The annular elution tube in our design proved suitable in preventing air bubbles from being trapped in the elution chamber; we never observed any during a great number of runs performed until now. The elution chamber has an invariable height of 3 mm. This value was found empirically to meet our demands. Under our experimental conditions a dye band of bromophenol blue with a width of about 4 mm at the end of the gel was eluted mainly in two fractions (6 ml) with traces of dye in t’he two adjacent fractions. In a t.est run using the SDS-Tris-buffer system ribonuclease (MW 13,700), and trypsin (MW 23,409#), and simultaneously cataIase (MW SO,OOO), and bovine serum albumine

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(MW 63,000) were separated almost without overlapping fractions, whereas in the case of lactate dehydrogenase (MW 36,000) and glycerol phosphate dehydrogenase (MW 40,000) only partial separation was achieved. The height of the elution chamber proved to be sufficient to tolerate gel swelling on the lower gel surface even after 48-72 hr run time. The hydrostatic equilibrium system is different from earlier designs. This is a consequence of the holes drilled into the upper part of the cooling finger and in that way having the electrophoresis buffer as the coolant. The rapid circulation of the electrophoresis buffer in the upper electrode chamber allows a drastic reduction in its space. By the overflows of the electrophoresis column the buffer height above the gel is brought to a level that allows the buffer in the elution cavity to act as manometer for the electrophoresis column. Even though hydrostatic equilibrium was already achieved in earlier designs the considerable pressure of the high buffer column above the gel made its maintenance crucial (in our design it is necessary only for very soft gels). Any manipulation during the run, for example, correction of the height of the elution chamber in case of gel swelling on the lower gel surface is a delicate operation. The large amount of buffer in the upper electrode chamber must be removed simultaneously with the buffer in the two manometers for the lower electrode vessel and the elution chamber, respectively. In our design the electrophoresis buffer in the upper electrode chamber may be discarded together with the elution buffer keeping perfect hydrostatic equilibrium at any time of the operation. The fitting of the cooling finger is different from earlier designs in two respects. The breakable glass part can be replaced easily, keeping all other parts intact. The connection to the upper electrode chamber is established without glass parts, which from our experience results in a safer fitting. By connection of the circulating cooling/electrophoresis buffer system to large buffer reservoirs the buffering capacity is considerably increased and the pH at both electrodes kept essentially constant. This is particularly important for long runs and using buffers with low ionic strength. Run times up to 72 hr have been tested. Using 25 liter reservoirs no exhaustion of the Tris-buffer system has been found. A circulation of buffer between both electrode vessels has already been introduced by Gordon et al. (7) but it was applicable to continuous buffer systems only. Our aim in introducing buffer circulation was to eliminate a separate cooling system and to reduce the buffer volume above the gel column for reasons discussed above. Finally, it should be mentioned that our design provides some improvements with respect to routine work:

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(i) By eliminating a separate cooling system, a membrane holder chamber, and separate manometers for the elution chamber and the lower electrode vessel, the number of operational steps is reduced; (ii) Because all outer parts except the easily exchangeable cooling finger are made from perspex or polyamide, the danger of breakage is minimized ; (iii) The total weight of the apparatus is remarkably reduced by displacing most of the electrophoresis buffer into reservoirs. This step especially facilitates the manipulation of the apparatus. We feel that with this design the technical aspect is no longer the limiting factor of the method. Now the major effort for improving separation should be directed to achieve the highest possible accuracy in maintaining the different bands horizontal and uniformly thick during their migration through the gel matrix. The implications of this point, which is an experimental rather than a technical one, have been discussed in detail recently (15). ACKNOWLEDGMENTS We want to thank Dr. U. Henning for his continuous interest and support of this work. We are indebted to Mr. E. Freiberg for performing the artwork and to Mr. M. Fordham for excellent workmanship and valuable suggestions in constructing the electrophoresis apparatus. The most valuable help of Drs. M. Achtman, J. Sulston, and A. Jesaitis in preparing the manuscript is gratefully appreciated. REFERENCES 1. RAYMOND, ORNSTEIN.

2.

S., AND L.,

AND

WEINTRAUB, DAVIS, B.

L. (1959) Science 130, 711. J. (1962) Disc Electrophoresis

(preprinted

by Dis-

tillation Products Industries,Rochester,N. Y.). 3.

H. R. (1971) Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, Walter de Gruyter, Berlin-New York. 4. GORDON, A. H. (1969) Electrophoresis of Proteins in Polyacrylamide and Starch Gel, North Holland Publishing Comp., Amsterdam-London. 5. CHRAMBACH. A., AND RODBARD, D. (1971) Scie/rce 172, 446. 6. JOVIN, T., CHRAMBACH, A., AND NAUGHTON, M. A. (1964) Anal. Biochem. Q, 351. 7. GORDOX. A. H., AND LOUIS, L. N. (1967) Anal. Biochem. 21, 190. 5. KALTSCHMIDT, E., AND WITTMANN, H. G. (1969) Anal. Biochem. 30, 132. 9. FLATGAARD, J. E., HOEHN, B., AND HENNING, U. (1971) Arch. Biochem. Biophys. 143, 461. 10. VOGEL, 0.. BEIKIRCH. H., MSLLER, H., AND HENNING. U. (1971) Eur. J. Biochem. 20, 169. 11. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4466. 12. VOGEL, 0.. AND HENNING, U. (1971) Eur. J. Biochem. 18, 163. 13. VOGEL, 0.. HOEHN, B., AND HENNING, U. (1972) Proc. Nat. Acd Sci. USA 69, 1615. 14. KAPADIA, G., AND CHRAMBA~H, A. (1972) Anal. Biochem. 48, 90, Appendix III. 15. 1,~NNEY, J., AND CHRAMBACH. A., AND RODBARD, D. (1971) Anal. B&hem. 40, 158. MAURER,