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A versatile apparatus for polyacrylamide and agarose gel electrophoresis in Plexiglas slab gel molds
ANALYTICAL
BIOCHEMISTRY
M),
A Versatile Apparatus Electrophoresis
624-632 (1978)
for Polyacrylamide and Agarose in Plexiglas Slab Gel Molds ROBERT
Department
of Microbiology. Farmington,
Gel
0. POYTON
University of Connecticut Connecticut 06032
Health
Center,
Received June 6. 1978 A simple vertical slab gel electrophoresis apparatus for analytical, preparative. and two-dimensional electrophoresis is described. The use of permanently sealed Plexiglas acrylic plastic slab gel molds which need to be sealed only at the bottom during gel formation, rather than the glass plate sandwich used in most previous designs, virtually eliminates leakage during gel formation and, in addition, permits the continuous monitoring with ultraviolet light of proteins and nucleic acids labeled with fluorescent dyes during electrophoresis. Results obtainable with this apparatus are equivalent to those achieved in other apparati which are more expensive to fabricate or purchase.
Electrophoresis in polyacrylamide or agarose slab gels has gained wide acceptance for both the analysis and preparation of proteins, polypeptides, nucleic acids, and oligonucleotides. During the past few years a number of designs for vertical slab gel electrophoresis units have been published (l- 15), some of which have served as prototypes for those which are commercially available. Most of these apparatuses fall into one of two categories. Some of them have slab gel molds with fixed glass plate walls and are modified versions of the apparatus originally described by Allen (l).’ Others have glass mold walls which are removable and are modifications of the units originally designed by Ackroyd (2-4), Reid and Bieleski (5-9),* Margolis and Kendrick (10,l 1),3 or Raymond4 (12-15). Designs of the first type are generally more difficult to construct and are limited to use with relatively low acrylamide gel concentrations (16). While this difficulty is avoided in designs of the second type, the use of removable glass plates has introduced a degree of inconvenience insofar as the seal between the plates is frequently incomplete and hence subject to leakage during gel preparation. i Available commercially from Ortec (Oak Ridge, Term.). 2 Available commercially from Blaircraft (Cold Spring Harbor, N. Y.), Bio-Rad Laboratories (Richmond, Calif.) and Hoeffer Scientific Instruments (San Francisco, Calif.). 3 Available commercially from Isolab (Akron, Ohio) and Pharmacia (Uppsala, Sweden). ’ Available commercially from E-C Apparatus Corporation (St. Petersberg, Florida). 0003-2697/78/0902-0624$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
624
VERTICAL
SLAB
GEL
ELECTROPHORESIS
625
While each design has some obvious merits and useful applications our experience suggests that many of these designs are either inconvenient to use (e.g., because of leakage during gel preparation or difficulties which arise from disrupting the gel surface while removing slot formers (see Ref. 7)), expensive to manufacture or purchase, or limited in application (e.g., due to the choice of material for construction or to the lack of provision for cooling of the slab gels during electrophoresis). Indeed, the use of glass walls for the gel molds in both of the above types of design imposes unnecessary limitations on their usefulness since it precludes the continuous monitoring of the electrophoretic migration of proteins and nucleic acids by fluorescent and ultraviolet techniques. The use of glass rather than Plexiglas walls in previously designed apparatuses seems to have been dictated in part by the observation that polyacrylamide gels adhere more tightly to glass than to Plexiglas (2,17,18) and the attendant fear held by earlier workers that the sample may seep between the gel and gel mold in gel molds made from acrylic sheets. While these reservations may be justified in some cases it is now clear that Plexiglas can be used effectiveiy for the walls of slab gel molds (19). In this paper we describe the design of a slab gel electrophoresis unit with fixed slab mold walls which uses to advantage both the optical properties of Plexiglas and its lowered affinity for polyacrylamide. METHODS The apparalus. The electrophoresis unit (Fig. IA) is constructed from clear Plexiglas (Plexiglas G from Rohm and Haas) and consists of: a lower vessel which houses the lower electrode and serves as a lower buffer chamber; an upper vessel which houses the upper electrode and two slab gel molds and which serves as an upper buffer chamber; and a vented top. While a number of different dimensions are permissible those which we find most useful are indicated in the legend to Fig. 1A. While this apparatus incorporates design elements used in a variety of other apparatus (e.g., Ref. 1,lO) it differs from previously published designs in that the slab gel molds are made entirely from Plexiglas and need to be sealed only at the bottom during gel polymerization (Fig. lB), a feature which effectively eliminates leakage during gel formation. Other useful design features include: (i) A bottom buffer chamber which surrounds the gel slab during electrophoresis and which holds a large enough volume of lower buffer to provide efficient cooling of the gels during electrophoresis. (ii) Walls of the slab gel molds and bottom buffer chamber which permit the penetration of ultraviolet light for the continuous monitoring of proteins and nucleic acids labeled with fluorescent dyes. Preparative polyacyfatnide gel electrophoresis. The upper vessel is
626
ROBERT
0. POYTON
FIG. 1. (A) Expanded view of slab gel electrophoresis apparatus. The lower and upper vessel walls and the top are made from %-in. thick Plexiglas while the baffles and slab gel mold walls are from %-in. Plexiglas. Lower vessel outside dimensions are 8 x 7 x 5 in. Upper vessel dimensions (exclusive of the slab gel molds) are 8 x 3% x 5 in. with one internal baffle measuring 8 x 1% in. and the other measuring 8 x XI in. The top dimensions are 8 x 5 in. The internal dimensions of the slab gel molds are 6 x 4% x l/s in. Upper and lower electrodes are made from No. 27.gauge platinum wire. (B) Casting stand with upper vessel in position for loading with gel solution. The casting stand consists of: a I2 x 12 x 1% in. Plexiglas base, two aluminum rods (12 x ‘/z in), two clamps machined from IV&in. Plexiglas, and a 9 x 5 x !G in. latex rubber sheet. (C) Exploded view of the casting stand clamp. (D) Schematic illustration of the positioning of dansylated markers and the undansylated sample for preparative electrophoresis. (E) Removal of the slab gel with the gel plunger (shown in amber Plexiglas) after electrophoresis.
VERTICAL
SLAB
GEL
627
ELECTROPHORESIS
mounted in the casting rack (Fig. 1B) on top of a %-in. latex rubber pad which is well greased with silicone vacuum grease (Dow Corning). A good seal is achieved by tightening down lightly and securing the upper vessel in place by the thumb screws (Figs. 1B and 1C). The SDS-polyacrylamide gel electrophoresis5 system used in our studies employs a discontinuous buffer system described in detail previously (20). Once mixed, the separating gel solution (60 ml per slab) is introduced into the slab gel mold by means of a syringe fitted with 1.5 mm-i.d. polyethylene tubing (Fig. 1B). The surface of the gel solution is then smoothed with a flat-tipped No. 24 gauge syringe needle (4 in. long) to assure equal adhesion to all sides, and the gel is overlayed with distilled water. The gel is then allowed to polymerize at room temperature (15-20 min), the water overlay is removed, and the upper stacking gel solution is introduced. For preparative electrophoresis with dansylated protein markers, slot-forming combs (made from 3-mm-thick Plexiglas or Teflon) are introduced into the stacking gel so that the bottom of each tooth is 0.5 cm above the lower gel (Fig. ID). The upper vessel is then removed from the casting rack and polymerized in front of a fluorescent light for 60 to 90 min. Once polymerized, the gel is overlayed with cathode buffer and the slot forming combs are removed gently. If only one slab gel is to be used the gel mold nearest to the 3?/1-in.-high baffle (Fig. IA) is used and the upper cathode buffer level is adjusted to be below the top of the 1%in.-high baffle. The sample in sample buffer is adjusted to 10% sucrose and O.OlO%, bromphenol blue and applied to the middle portion of each slab bracketed on both sides by sample which had been dansylated as described by Taibot and Yphantis (21). Electrophoresis is initiated at 1.5mA per slab and continued until the tracking dye, bromphenol blue, has migrated through the stacking gel (about 60 min): the current is then increased to 40 mA per slab and electrophoresis is continued until the desired resolution, as judged by fluorescence (366 nm peak output) has been achieved. Once electrophoresis is completed the gels are loosened by rimming with water from a 6-in.-long flat-tipped No. 22 gauge needle and removed from the gel mold by gently pushing (Fig. IE) with the gel plunger (dimensions, 6 x 7 x ‘/s in.). Gel slices containing the desired protein are removed from the rest of the gel slab using the fluorescent samples on each edge of the gel slab as markers. Protein is then electroeluted into a dialysis bag as described previously (20). At~dytical polycrcrylamide gel electrophoresis. For making analytical polyacrylamide gels (0.75 mm thick) two glass plates (6 x 4% x 3164in.) separated by 0.75-mm-thick Plexiglas spacers are inserted into the slab gel molds and the gels are prepared as described above for preparative polyacrylamide electrophoresis except that the amount of separating gel solution is reduced to 20 ml per slab. Following polymerization of the 5 Abbreviations sodium dodecyl
used: sulfate.
SDS-PAGE,
polyacrylamide
gel electrophoresis
in the presence
of
628
ROBERT
0. POYTON
separating gel enough stacking gel solution is added to fill the slab gel mold, multiple slot forming combs are inserted, and the gel is polymerized in front of a fluorescent light as above. Samples are added as for preparative gel electrophoresis and electrophoresis is performed at 8 mA/slab while the sample is in the stacking gel and 20 mA/slab while the sample is in the resolving gel. When the tracking dye reaches the bottom of the gel, electrophoresis is terminated and the glass plate-gel sandwich is removed with the gel plunger as described in the previous section. The two Plexiglas spacers are then removed from the glass plate-gel sandwich and the plates are popped apart by inserting a narrow strip of l/32-in.-thick Plexiglas and twisting. The gels are then fixed in 50% trichloroacetic acid for 30 min, are stained in 0.1% Coomassie blue (R250)-50% trichloroacetic acid, are destained in 7.5% acetic acid overnight, are dried on a Hoeffer gel drier and are subjected to autoradiography on Kodak SB-5 film or fluorography (22) on Kodak X-R-5 film. Two-dimensional electrophoresis. For two-dimensional analysis, samples are first subjected to isoelectric focusing in 3-mm-thick slab gels and then to sodium dodecyl sulfate polyacrylamide gel electrophoresis in 0.75mm-thick slab gels. Isoelectric focusing is performed in g-cm-long polyacrylamide gels containing 4.0% acrylamide, 0.141% N,N’-methylenebisacrylamide, 8 M urea, 2% Triton X-100, 2% ampholine (LKB, pH range 3-lo), 0.0005% riboflavin, and 0.0075% ammonium persulfate. Gels are made, polymerized, and run as described by Finlayson and Chrambach (23). Isoelectric focusing is performed at 34 V/cm for 16 to 18 h. In order to prevent the gels from slipping out of the gel molds during focusing, polyethylene foam sponges saturated with anode buffer (23) are wedged between the bottom of the gel molds and the bottom of the lower vessel.
Slab
Gel Mold
Walls
FIG. 2. Illustration of the apparatus set up for two-dimensional section of slab gel mold with the glass plate sandwich in place.
electrophoresis. (B) Front view
(A) Cross of same.
VERTICAL
SLAB
GEL
629
ELECTROPHORESIS
The first dimension can also be run in glass tubes (3 mm i.d.) (24). The first dimension slab gels are cut into 3-mm-wide strips, incubated in 1% SDS- 10 mM NaPO, (pH 7.0)-6 M urea- 1% P-mercaptoethanol at 37°C for 60 min, and then laid across the top of a stacking gel prepared on top of a 0.75mmthick analytical polyacrylamide gel (Fig. 2) prepared as described above. The first dimension gel is fixed in place with a solution of 1% agarose in 10 mM NaPO,, pH 7.0, heated to 80°C. One-slot combs for marker proteins are inserted and the agarose is allowed to harden (5-10 min). Then the marker protein samples in sample buffer are added, and the upper buffer is made 0.0001% bromphenol blue. Electrophoresis is carried out as described above for analytical gels. Agarosr gel electrophoresis. For preparing agarose gels (3 mm thick) the upper vessel is mounted in the casting rack as described above. The agarose solution made 0.5 to 1.5% in E buffer (7) and containing 0.5 pg of ethidium bromide per ml is adjusted to 60°C and added to the top of the gel molds as described above. Teflon slot-forming combs, one-half the thickness of the gel, are added and the agarose is allowed to harden (10 min). The use of slot-forming combs, which are thinner than the gel itself greatly reduces the chance of breaking the agarose slots upon re-
PH 8
7
6
5
M.W. -1 (K)
4
- 70 -52 -32
FIG. 3. Two-dimensional electrophoretic patterns of yeast mitochondrial membranes. About 50 pg of membrane protein was applied to the first dimension isoelectric focusing gel. The acrylamide monomer concentration in the second dimension was 12%. The marker slots at the left were loaded with different amounts (20 and 60 pg of protein) of the same membrane preparation which was electrophoresed in both dimensions.
630
ROBERT
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moval of the slot formers. If thicker slots are desired an alternative method for removing the slot-forming combs is used. After the agarose has hardened the gel is pushed partially out of the top of the gel mold with the plunger applied to the bottom. The slot-forming combs are then removed and the gel is allowed to slide back down to the bottom of the slab gel mold. To prevent the gels from sliding out of the slab gel mold a piece of cheesecloth held in place by a rubber band is affixed to the bottom of the gel mold. Gels are then loaded and run as described previously (7) and the electrophoretic migration of each sample is monitored continually by observing ethidium bromide fluorescence after excitation with a focused short-wavelength ultraviolet light. Special procedures. Since the slab gel molds are made from Plexiglas special precautions must be observed in cleaning them. In our hands the most effective way to clean the slab gel molds are to first wash them in a 40°C solution of 1% SDS (for SDS-gels) or 1% Triton X-100 (native gels), then rinse them by immersion in 70% ethanol followed by several washes with distilled water. These washes effectively remove all traces of detergent. Silicone grease is easily removed from the inside of the slab molds by washing with a cotton swab in 70% ethanol. RESULTS AND DISCUSSION Since its original design 5 years ago the apparatus described in this paper has proven useful for a variety of applications including: the isolation of yeast cytochrome c oxidase on Triton X-100 containing polyacrylamide gels (Poyton, R. O., unpublished observations) and cytochrome c oxidase subunits by preparative SDS-PAGE (20), isoelectric focusing of active cytochrome c oxidase, inactive cytochrome c oxidase subunits, and total mitochondrial membrane proteins (George, D. and Poyton, R. O., unpublished observations); two-dimensional electrophoresis employing isoelectric focusing and SDS-PAGE (Fig. 3 and George, D. and Poyton, R. 0.) unpublished observations); orthacryl two-dimensional SDS-PAGE (25); analytical and preparative SDS-PAGE of polypeptides from bacterial (26; H. Wu, personal communication) and synaptic membranes (L. Rothfield, personal communication); and agarose gel analysis of restriction fragments in DNA digests (Fig. 4 and C. Tibbetts, personal communication). Taken together these studies attest to the versatility of this design and have indicated that it can be used at polyacrylamide gel concentrations from 4 to 22.5% T,” agarose contrations above 0.5%, pH values from 3.5 to 10, and temperatures from 4 to 40°C. It has proven to be particularly convenient for two-dimensional electrophoresis since it does not require specially cut or slotted glass plates (cf. 24). Some of the most useful design features of this apparatus are the use of gel molds which are: permanently fixed; and made from Plexiglas. As Ii The nomenclature to are 3% C.
of Hjerten
(29) is used to describe
gel composition.
Gels
referred
VERTICAL
SLAB
GEL
ELECTROPHORESIS
631
FIG. 4. Use of agarose gel electrophoresis to titrate sac 1 restriction endonuclease activity on adenovirus 7 DNA. Conditions for digestion and photographing the gel by uv illumination were as described previously (28). Each slot contained 0.07 pg of adenovirus 7 DNA. Undigested DNA is present in slots 1 and 8. Digests run in slots 2 through 7 used increasing amounts of restriction enzyme.
with previous designs the use of permanently fixed gel mold walls effectively eliminates leakage during gel preparation. The use of Plexiglas rather than glass for the gel mold wall represents a significant departure from previous designs and greatly extends the versatility of the unit since it permits the continuous monitoring of electrophoresis by uv and fluorescent techniques (21,27) without necessitating the use of expensive opticalquality quartz plates (27). In addition, the use of Plexiglas rather than glass for the gel mold wall permits the easy removal of the slab gel after electrophoresis since polyacrylamide possess a lower affinity for Plexiglas than glass. It is worth noting that nearly all previous designs appear to have carefully avoided the use of Plexiglas in slab gel walls for fear that the sample would seep between the gel and slab wall (2,17,18), that the Plexiglas might be distorted by heat emitted during polyacrylamide polymerization (9), or that the heat transfer across Plexiglas might be insufficient to dissipate ohmic heat generated during electrophoresis. None of these reservations appears justified when using this apparatus for either polyacrylamide gel isoelectric focusing in gels of 4 to 7.5% T or SDSPAGE in gels of 4 to 22% T at temperatures ranging from 4 to 40°C. However, as with apparati with glass mold walls, sample seepage is a problem during agarose gel electrophoresis on gels of 0.8% agarose or less. We find that this problem is most easily avoided by using slot-forming combs which are one-half the thickness of slab gel and hence running the sample in the
632
ROBERT 0. POYTON
middle portion of the gel, away from the gel mold walls. It is worth emphasizing that the use of Plexiglas mold walls demands that special care be taken in cleaning the gel molds and in removing the gels from the unit so as not to scratch the gel mold wall. While these are clearly disadvantages they are offset by the convenience of use, versatility, and low cost of fabrication offered by Plexiglas. ACKNOWLEDGMENTS This work was supported in part by grants from the National Institute of Health (GM 21800) and the American Heart Association (75-779). The author thanks Dr. Clark Tibbetts for kindly providing the adenovirus DNA used in this study and for relating his experience pertaining to agarose gel electrophoresis. and Drs. L. Rothfield, H. Wu. and J. Foulds for their comments on the applicability of these apparati to the analysis and preparation of a number of membrane proteins. In addition, the author wishes to thank Mr. Ernest Brown and John Soracchi of the Technical Services Center (University of Connecticut, Storm Campus) for their excellent cooperation and workmanship during the construction of these apparati.
REFERENCES 1. Allen, R. C., and Moore, D. J. (1966) Anal. Biochem. 16, 457-465. 2. Akroyd, P. (1967) And. Biochem. 19. 399-410. 3. Rowe. J. (1973) Clin. Chem. Aciu 47. 63-68. 4. Abadi, D. M. (1969) Clin. Chem. 15, 35-41. 5. Reid, M. S., and Bieleski, R. L. (1968) Anal. Biochem. 22, 374-381. 6. Studier, F. W. (1973) J. Molec. Biol. 79, 237-248. 7. Sugden, B.. DeTroy. B., Roberts, R. J.. and Sambrook, J. (1975)
And.
Biochem.
68, 36-46. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
25. 26. 27. 28. 29.
Amos, W. B. (1976)Anal. Biochem. 70, 612-615. Madjar, J.-J., Arpin, M., and Reboud, J-P. (1977) Anal. Biochem. 83, 304-310. Margolis, J., and Kenrick, K. G. (1968) Anal. Biochem. 25, 347-362. Hoffman, W. L., and Ban, J. 1976. Prep. Biochem. 6, 13-26. Raymond, S. 1962. C/in. Chem. 8, 455-460. Woodworth, R. C., and Clark, L. G. (1967) Anal. Biochem. 18, 295-304. Roberts, R. M., and Jones, J. S. (1972) Anal. B&hem. 49, 592-597. Yeger, H., and Freedman, M. (1975) Anal. Biochem. 64, 450-457. Ortec Application Note #AN 32A, p. 20. Blattner, D. P. (1969) And. Biochem. 27, 73-76. Stuyvesant, V. W. (1967) Nature (London) 214, 405-407. Sauaia, H., and Laicine, E. M. (1977) Anal. Biochem. 80, 125-132. Poyton, R. O., and Schatz, G. (1975) J. Biol. Chem. 250, 752-761. Talbot, D. N., and Yphantis, D. A. (1971) And. Biochem. 44, 246-253. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. Finlayson, G. R. and Chrambach, A. (1971) Anal. Biochem. 40, 292-311. O’Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021. Poyton, R. O., McKemmie, E., and -Nascimento. C. G. (1978) J. Biol. Chem. in press. Chai, T. J., and Foulds, J. (1977) Biochim. Biophys. Acta 493, 210-215. Eisinger, J. (1971) Biochem. Biophys. Res. Commun. 44, 1135-1142. Tibbetts, C. (1977) J. Viral. 24. 564-579. Hjerten, S. (1962) Arch. Biochem. Biophys. 1 (Suppl. lo), 147-151.
253,
BIOCHEMISTRY
M),
A Versatile Apparatus Electrophoresis
624-632 (1978)
for Polyacrylamide and Agarose in Plexiglas Slab Gel Molds ROBERT
Department
of Microbiology. Farmington,
Gel
0. POYTON
University of Connecticut Connecticut 06032
Health
Center,
Received June 6. 1978 A simple vertical slab gel electrophoresis apparatus for analytical, preparative. and two-dimensional electrophoresis is described. The use of permanently sealed Plexiglas acrylic plastic slab gel molds which need to be sealed only at the bottom during gel formation, rather than the glass plate sandwich used in most previous designs, virtually eliminates leakage during gel formation and, in addition, permits the continuous monitoring with ultraviolet light of proteins and nucleic acids labeled with fluorescent dyes during electrophoresis. Results obtainable with this apparatus are equivalent to those achieved in other apparati which are more expensive to fabricate or purchase.
Electrophoresis in polyacrylamide or agarose slab gels has gained wide acceptance for both the analysis and preparation of proteins, polypeptides, nucleic acids, and oligonucleotides. During the past few years a number of designs for vertical slab gel electrophoresis units have been published (l- 15), some of which have served as prototypes for those which are commercially available. Most of these apparatuses fall into one of two categories. Some of them have slab gel molds with fixed glass plate walls and are modified versions of the apparatus originally described by Allen (l).’ Others have glass mold walls which are removable and are modifications of the units originally designed by Ackroyd (2-4), Reid and Bieleski (5-9),* Margolis and Kendrick (10,l 1),3 or Raymond4 (12-15). Designs of the first type are generally more difficult to construct and are limited to use with relatively low acrylamide gel concentrations (16). While this difficulty is avoided in designs of the second type, the use of removable glass plates has introduced a degree of inconvenience insofar as the seal between the plates is frequently incomplete and hence subject to leakage during gel preparation. i Available commercially from Ortec (Oak Ridge, Term.). 2 Available commercially from Blaircraft (Cold Spring Harbor, N. Y.), Bio-Rad Laboratories (Richmond, Calif.) and Hoeffer Scientific Instruments (San Francisco, Calif.). 3 Available commercially from Isolab (Akron, Ohio) and Pharmacia (Uppsala, Sweden). ’ Available commercially from E-C Apparatus Corporation (St. Petersberg, Florida). 0003-2697/78/0902-0624$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
624
VERTICAL
SLAB
GEL
ELECTROPHORESIS
625
While each design has some obvious merits and useful applications our experience suggests that many of these designs are either inconvenient to use (e.g., because of leakage during gel preparation or difficulties which arise from disrupting the gel surface while removing slot formers (see Ref. 7)), expensive to manufacture or purchase, or limited in application (e.g., due to the choice of material for construction or to the lack of provision for cooling of the slab gels during electrophoresis). Indeed, the use of glass walls for the gel molds in both of the above types of design imposes unnecessary limitations on their usefulness since it precludes the continuous monitoring of the electrophoretic migration of proteins and nucleic acids by fluorescent and ultraviolet techniques. The use of glass rather than Plexiglas walls in previously designed apparatuses seems to have been dictated in part by the observation that polyacrylamide gels adhere more tightly to glass than to Plexiglas (2,17,18) and the attendant fear held by earlier workers that the sample may seep between the gel and gel mold in gel molds made from acrylic sheets. While these reservations may be justified in some cases it is now clear that Plexiglas can be used effectiveiy for the walls of slab gel molds (19). In this paper we describe the design of a slab gel electrophoresis unit with fixed slab mold walls which uses to advantage both the optical properties of Plexiglas and its lowered affinity for polyacrylamide. METHODS The apparalus. The electrophoresis unit (Fig. IA) is constructed from clear Plexiglas (Plexiglas G from Rohm and Haas) and consists of: a lower vessel which houses the lower electrode and serves as a lower buffer chamber; an upper vessel which houses the upper electrode and two slab gel molds and which serves as an upper buffer chamber; and a vented top. While a number of different dimensions are permissible those which we find most useful are indicated in the legend to Fig. 1A. While this apparatus incorporates design elements used in a variety of other apparatus (e.g., Ref. 1,lO) it differs from previously published designs in that the slab gel molds are made entirely from Plexiglas and need to be sealed only at the bottom during gel polymerization (Fig. lB), a feature which effectively eliminates leakage during gel formation. Other useful design features include: (i) A bottom buffer chamber which surrounds the gel slab during electrophoresis and which holds a large enough volume of lower buffer to provide efficient cooling of the gels during electrophoresis. (ii) Walls of the slab gel molds and bottom buffer chamber which permit the penetration of ultraviolet light for the continuous monitoring of proteins and nucleic acids labeled with fluorescent dyes. Preparative polyacyfatnide gel electrophoresis. The upper vessel is
626
ROBERT
0. POYTON
FIG. 1. (A) Expanded view of slab gel electrophoresis apparatus. The lower and upper vessel walls and the top are made from %-in. thick Plexiglas while the baffles and slab gel mold walls are from %-in. Plexiglas. Lower vessel outside dimensions are 8 x 7 x 5 in. Upper vessel dimensions (exclusive of the slab gel molds) are 8 x 3% x 5 in. with one internal baffle measuring 8 x 1% in. and the other measuring 8 x XI in. The top dimensions are 8 x 5 in. The internal dimensions of the slab gel molds are 6 x 4% x l/s in. Upper and lower electrodes are made from No. 27.gauge platinum wire. (B) Casting stand with upper vessel in position for loading with gel solution. The casting stand consists of: a I2 x 12 x 1% in. Plexiglas base, two aluminum rods (12 x ‘/z in), two clamps machined from IV&in. Plexiglas, and a 9 x 5 x !G in. latex rubber sheet. (C) Exploded view of the casting stand clamp. (D) Schematic illustration of the positioning of dansylated markers and the undansylated sample for preparative electrophoresis. (E) Removal of the slab gel with the gel plunger (shown in amber Plexiglas) after electrophoresis.
VERTICAL
SLAB
GEL
627
ELECTROPHORESIS
mounted in the casting rack (Fig. 1B) on top of a %-in. latex rubber pad which is well greased with silicone vacuum grease (Dow Corning). A good seal is achieved by tightening down lightly and securing the upper vessel in place by the thumb screws (Figs. 1B and 1C). The SDS-polyacrylamide gel electrophoresis5 system used in our studies employs a discontinuous buffer system described in detail previously (20). Once mixed, the separating gel solution (60 ml per slab) is introduced into the slab gel mold by means of a syringe fitted with 1.5 mm-i.d. polyethylene tubing (Fig. 1B). The surface of the gel solution is then smoothed with a flat-tipped No. 24 gauge syringe needle (4 in. long) to assure equal adhesion to all sides, and the gel is overlayed with distilled water. The gel is then allowed to polymerize at room temperature (15-20 min), the water overlay is removed, and the upper stacking gel solution is introduced. For preparative electrophoresis with dansylated protein markers, slot-forming combs (made from 3-mm-thick Plexiglas or Teflon) are introduced into the stacking gel so that the bottom of each tooth is 0.5 cm above the lower gel (Fig. ID). The upper vessel is then removed from the casting rack and polymerized in front of a fluorescent light for 60 to 90 min. Once polymerized, the gel is overlayed with cathode buffer and the slot forming combs are removed gently. If only one slab gel is to be used the gel mold nearest to the 3?/1-in.-high baffle (Fig. IA) is used and the upper cathode buffer level is adjusted to be below the top of the 1%in.-high baffle. The sample in sample buffer is adjusted to 10% sucrose and O.OlO%, bromphenol blue and applied to the middle portion of each slab bracketed on both sides by sample which had been dansylated as described by Taibot and Yphantis (21). Electrophoresis is initiated at 1.5mA per slab and continued until the tracking dye, bromphenol blue, has migrated through the stacking gel (about 60 min): the current is then increased to 40 mA per slab and electrophoresis is continued until the desired resolution, as judged by fluorescence (366 nm peak output) has been achieved. Once electrophoresis is completed the gels are loosened by rimming with water from a 6-in.-long flat-tipped No. 22 gauge needle and removed from the gel mold by gently pushing (Fig. IE) with the gel plunger (dimensions, 6 x 7 x ‘/s in.). Gel slices containing the desired protein are removed from the rest of the gel slab using the fluorescent samples on each edge of the gel slab as markers. Protein is then electroeluted into a dialysis bag as described previously (20). At~dytical polycrcrylamide gel electrophoresis. For making analytical polyacrylamide gels (0.75 mm thick) two glass plates (6 x 4% x 3164in.) separated by 0.75-mm-thick Plexiglas spacers are inserted into the slab gel molds and the gels are prepared as described above for preparative polyacrylamide electrophoresis except that the amount of separating gel solution is reduced to 20 ml per slab. Following polymerization of the 5 Abbreviations sodium dodecyl
used: sulfate.
SDS-PAGE,
polyacrylamide
gel electrophoresis
in the presence
of
628
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0. POYTON
separating gel enough stacking gel solution is added to fill the slab gel mold, multiple slot forming combs are inserted, and the gel is polymerized in front of a fluorescent light as above. Samples are added as for preparative gel electrophoresis and electrophoresis is performed at 8 mA/slab while the sample is in the stacking gel and 20 mA/slab while the sample is in the resolving gel. When the tracking dye reaches the bottom of the gel, electrophoresis is terminated and the glass plate-gel sandwich is removed with the gel plunger as described in the previous section. The two Plexiglas spacers are then removed from the glass plate-gel sandwich and the plates are popped apart by inserting a narrow strip of l/32-in.-thick Plexiglas and twisting. The gels are then fixed in 50% trichloroacetic acid for 30 min, are stained in 0.1% Coomassie blue (R250)-50% trichloroacetic acid, are destained in 7.5% acetic acid overnight, are dried on a Hoeffer gel drier and are subjected to autoradiography on Kodak SB-5 film or fluorography (22) on Kodak X-R-5 film. Two-dimensional electrophoresis. For two-dimensional analysis, samples are first subjected to isoelectric focusing in 3-mm-thick slab gels and then to sodium dodecyl sulfate polyacrylamide gel electrophoresis in 0.75mm-thick slab gels. Isoelectric focusing is performed in g-cm-long polyacrylamide gels containing 4.0% acrylamide, 0.141% N,N’-methylenebisacrylamide, 8 M urea, 2% Triton X-100, 2% ampholine (LKB, pH range 3-lo), 0.0005% riboflavin, and 0.0075% ammonium persulfate. Gels are made, polymerized, and run as described by Finlayson and Chrambach (23). Isoelectric focusing is performed at 34 V/cm for 16 to 18 h. In order to prevent the gels from slipping out of the gel molds during focusing, polyethylene foam sponges saturated with anode buffer (23) are wedged between the bottom of the gel molds and the bottom of the lower vessel.
Slab
Gel Mold
Walls
FIG. 2. Illustration of the apparatus set up for two-dimensional section of slab gel mold with the glass plate sandwich in place.
electrophoresis. (B) Front view
(A) Cross of same.
VERTICAL
SLAB
GEL
629
ELECTROPHORESIS
The first dimension can also be run in glass tubes (3 mm i.d.) (24). The first dimension slab gels are cut into 3-mm-wide strips, incubated in 1% SDS- 10 mM NaPO, (pH 7.0)-6 M urea- 1% P-mercaptoethanol at 37°C for 60 min, and then laid across the top of a stacking gel prepared on top of a 0.75mmthick analytical polyacrylamide gel (Fig. 2) prepared as described above. The first dimension gel is fixed in place with a solution of 1% agarose in 10 mM NaPO,, pH 7.0, heated to 80°C. One-slot combs for marker proteins are inserted and the agarose is allowed to harden (5-10 min). Then the marker protein samples in sample buffer are added, and the upper buffer is made 0.0001% bromphenol blue. Electrophoresis is carried out as described above for analytical gels. Agarosr gel electrophoresis. For preparing agarose gels (3 mm thick) the upper vessel is mounted in the casting rack as described above. The agarose solution made 0.5 to 1.5% in E buffer (7) and containing 0.5 pg of ethidium bromide per ml is adjusted to 60°C and added to the top of the gel molds as described above. Teflon slot-forming combs, one-half the thickness of the gel, are added and the agarose is allowed to harden (10 min). The use of slot-forming combs, which are thinner than the gel itself greatly reduces the chance of breaking the agarose slots upon re-
PH 8
7
6
5
M.W. -1 (K)
4
- 70 -52 -32
FIG. 3. Two-dimensional electrophoretic patterns of yeast mitochondrial membranes. About 50 pg of membrane protein was applied to the first dimension isoelectric focusing gel. The acrylamide monomer concentration in the second dimension was 12%. The marker slots at the left were loaded with different amounts (20 and 60 pg of protein) of the same membrane preparation which was electrophoresed in both dimensions.
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moval of the slot formers. If thicker slots are desired an alternative method for removing the slot-forming combs is used. After the agarose has hardened the gel is pushed partially out of the top of the gel mold with the plunger applied to the bottom. The slot-forming combs are then removed and the gel is allowed to slide back down to the bottom of the slab gel mold. To prevent the gels from sliding out of the slab gel mold a piece of cheesecloth held in place by a rubber band is affixed to the bottom of the gel mold. Gels are then loaded and run as described previously (7) and the electrophoretic migration of each sample is monitored continually by observing ethidium bromide fluorescence after excitation with a focused short-wavelength ultraviolet light. Special procedures. Since the slab gel molds are made from Plexiglas special precautions must be observed in cleaning them. In our hands the most effective way to clean the slab gel molds are to first wash them in a 40°C solution of 1% SDS (for SDS-gels) or 1% Triton X-100 (native gels), then rinse them by immersion in 70% ethanol followed by several washes with distilled water. These washes effectively remove all traces of detergent. Silicone grease is easily removed from the inside of the slab molds by washing with a cotton swab in 70% ethanol. RESULTS AND DISCUSSION Since its original design 5 years ago the apparatus described in this paper has proven useful for a variety of applications including: the isolation of yeast cytochrome c oxidase on Triton X-100 containing polyacrylamide gels (Poyton, R. O., unpublished observations) and cytochrome c oxidase subunits by preparative SDS-PAGE (20), isoelectric focusing of active cytochrome c oxidase, inactive cytochrome c oxidase subunits, and total mitochondrial membrane proteins (George, D. and Poyton, R. O., unpublished observations); two-dimensional electrophoresis employing isoelectric focusing and SDS-PAGE (Fig. 3 and George, D. and Poyton, R. 0.) unpublished observations); orthacryl two-dimensional SDS-PAGE (25); analytical and preparative SDS-PAGE of polypeptides from bacterial (26; H. Wu, personal communication) and synaptic membranes (L. Rothfield, personal communication); and agarose gel analysis of restriction fragments in DNA digests (Fig. 4 and C. Tibbetts, personal communication). Taken together these studies attest to the versatility of this design and have indicated that it can be used at polyacrylamide gel concentrations from 4 to 22.5% T,” agarose contrations above 0.5%, pH values from 3.5 to 10, and temperatures from 4 to 40°C. It has proven to be particularly convenient for two-dimensional electrophoresis since it does not require specially cut or slotted glass plates (cf. 24). Some of the most useful design features of this apparatus are the use of gel molds which are: permanently fixed; and made from Plexiglas. As Ii The nomenclature to are 3% C.
of Hjerten
(29) is used to describe
gel composition.
Gels
referred
VERTICAL
SLAB
GEL
ELECTROPHORESIS
631
FIG. 4. Use of agarose gel electrophoresis to titrate sac 1 restriction endonuclease activity on adenovirus 7 DNA. Conditions for digestion and photographing the gel by uv illumination were as described previously (28). Each slot contained 0.07 pg of adenovirus 7 DNA. Undigested DNA is present in slots 1 and 8. Digests run in slots 2 through 7 used increasing amounts of restriction enzyme.
with previous designs the use of permanently fixed gel mold walls effectively eliminates leakage during gel preparation. The use of Plexiglas rather than glass for the gel mold wall represents a significant departure from previous designs and greatly extends the versatility of the unit since it permits the continuous monitoring of electrophoresis by uv and fluorescent techniques (21,27) without necessitating the use of expensive opticalquality quartz plates (27). In addition, the use of Plexiglas rather than glass for the gel mold wall permits the easy removal of the slab gel after electrophoresis since polyacrylamide possess a lower affinity for Plexiglas than glass. It is worth noting that nearly all previous designs appear to have carefully avoided the use of Plexiglas in slab gel walls for fear that the sample would seep between the gel and slab wall (2,17,18), that the Plexiglas might be distorted by heat emitted during polyacrylamide polymerization (9), or that the heat transfer across Plexiglas might be insufficient to dissipate ohmic heat generated during electrophoresis. None of these reservations appears justified when using this apparatus for either polyacrylamide gel isoelectric focusing in gels of 4 to 7.5% T or SDSPAGE in gels of 4 to 22% T at temperatures ranging from 4 to 40°C. However, as with apparati with glass mold walls, sample seepage is a problem during agarose gel electrophoresis on gels of 0.8% agarose or less. We find that this problem is most easily avoided by using slot-forming combs which are one-half the thickness of slab gel and hence running the sample in the
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middle portion of the gel, away from the gel mold walls. It is worth emphasizing that the use of Plexiglas mold walls demands that special care be taken in cleaning the gel molds and in removing the gels from the unit so as not to scratch the gel mold wall. While these are clearly disadvantages they are offset by the convenience of use, versatility, and low cost of fabrication offered by Plexiglas. ACKNOWLEDGMENTS This work was supported in part by grants from the National Institute of Health (GM 21800) and the American Heart Association (75-779). The author thanks Dr. Clark Tibbetts for kindly providing the adenovirus DNA used in this study and for relating his experience pertaining to agarose gel electrophoresis. and Drs. L. Rothfield, H. Wu. and J. Foulds for their comments on the applicability of these apparati to the analysis and preparation of a number of membrane proteins. In addition, the author wishes to thank Mr. Ernest Brown and John Soracchi of the Technical Services Center (University of Connecticut, Storm Campus) for their excellent cooperation and workmanship during the construction of these apparati.
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253,