ANALYTICAL
BIOCHEMISTRY
Highly
84,
354-360 (1978)
Simplified Analytical or Preparative Gel Electrophoresis
Slab
J. P. KERCKAERT Institut de Recherches sur le Cancer de Liile (Institut Jules Driessens) et Uniti no 124 INSERM, BP 3567. 59020 Lille Ct5de.x.France Received November 24, 1976; accepted August 29, 1977 A simple, rapid, and efficient procedure has been developed for performing analytical or preparative slab gel electrophoresis using only common laboratory materials which can be assembled without special tools or equipment. From one to four gel slabs of variable size can be made between glass plates embedded inside a watertight, supple plastic bag which is then used as the upper electrode buffer chamber. This technique has been applied, with different electrophoretic systems, for both the analysis and isolation of serum proteins and rat liver histones.
During the past 10 years a number of apparatus have been designed for electrophoresis on polyacrylamide gel slabs (l- 14). However, for many workers the construction or the use of such devices may present some difficulties, especially in obtaining a liquid-tight gel mold. This often requires precise machining or the use of sealing material, grease (l-5,7,8), melted agar (7,13), rubber gaskets (6,10-12), adhesive tape (2,9,14), or polyacrylamide plugs (10). Further, in connecting the gel with the electrode buffer, one often needs to install a filter paper bridge (5) or to mount and clamp tightly the gel slab on the electrophoresis apparatus (l-4,6-8,11,13,14). To eliminate all these operations, I have developed a very simple, inexpensive device which can be set up rapidly and which gives excellent results for both analytical and preparative experiments. Simplification is obtained by using a plastic bag, first to maintain the acrylamide solution in the polymerization cell and, second, to serve as the upper electrode chamber. In contrast to other simple electrophoretic devices, the procedure described here does not require the machining of special equipment. Only common materials are needed: glass plates, glass spacers, a plastic bag, rubber bands, clothes pins, a beaker, and a power supply with platinium electrodes. In addition, the flexibility of this procedure allows the use of various sizes of gel slabs with up to four slabs per experiment. 0003-2697178/0842-0354$02.00/O Copyright All rights
Q 1978 by Academic Press, Inc. of reproduction in any form reserved.
354
SIMPLIFIED
SLAB
GEL
355
ELECTROPHORESIS
METHODS Preparative procedure. The operating procedure is schematically divided into six steps as illustrated in Fig. 1. (i) To form the gel chamber, two glass plates (A) and two glass spacers (B) are placed together without any sealing device in a plastic bag (C). The spacers can be easily held in place by ensuring a gentle pressure on the glass plates with one’s fingers. The height of the bag is about two times that of the glass plates. Plastic bags of appropriate sizes can be made with the aid of a plastic sealer (Soud Sac, Calor, France) from bulk plastic sheets commonly used for book covering. The bag must be transparent, supple but tear resistant, watertight, and clean. Generally, 20 x 20-cm plates with 0.5-cm-thick glass spacers (0.5 x 1.5 x 20 cm) are used. Up to four gel slabs can be polymerized simultaneously using five glass plates separated by four pairs of spacers. (ii) The bag is folded if too large and is pressed against the gel chamber by two outside glass plates (D). The assembly is secured with
El
El
H-
FIG. 1. Schematic representation of preparative slab gel electrophoresis. Glass plates; (B) glass spacers; (C) supple watertight plastic bag; (E) rubber acrylamide solution; (G) isobutanol overlay: (H) cutting off the bottom of the bag; to elevate the assembly; (K) sample syringe; (L) electrode buffer; (M) platinium Explanations of the different steps are detailed under Methods.
(A and D) bands; (F) (J) wedges electrodes.
356
J. P. KERCKAERT
rubber bands (E). Plates and spacers can then be readjusted, if necessary, inside the bag. (iii) The gel monomer solution (F) is poured into the bag to about 5 cm from the top of the plates. The gel is then overlaid with the aid of a Pasteur pipet with water-saturated isobutyl alcohol (G) to produce a flat surface and the gel is allowed to polymerize. (iv) After polymerization the surface of the gel is rinsed with distilled water and the bottom of the bag is cut off(H). (v) The assembly is placed vertically on two small (0.5 cm thick) glass or acrylic wedges (J) in a beaker or other tank. The surface of the gel must be horizontal to avoid skewed bands. The tank is used as the lower electrode chamber and it must be high enough to allow for attachment of the plastic bag to the walls of the chamber with clothes pins or paper clips. Electrode buffer is then poured in the tank and in the bag until the levels in these two compartments are the same. This eliminates pressure on the plastic sack and gives it a more suitable shape and a larger volume. Samples (1 to 5 ml per slab, containing 10 to 100 mg of proteins) made denser with 20% (w/v) glycerol or sucrose or with 8 M urea, are layered on the gel surface with the aid of a syringe fitted with a short piece of tubing (K). A trace of bromophenol blue (for alkaline gels) or pyronin red (for acidic gels) is added to the sample to visualize the layering. (vi) Platinium electrodes (M) are placed in the two buffer chambers and connected to a power supply. Up to 100 mA/slab with 80-150 Volts can be applied without excessive heating of the gel. Generally electrophoresis is carried out overnight and, if necessary, to avoid changes in buffer composition, an aquarium centrifuge pump can be installed as illustrated in Fig. 2 to allow buffer circulation from the lower to the upper electrode chamber. After completion of electrophoresis, the bag and the glass plates are disassembled. Two lateral guide strips of gel (1 cm large) are cut out, stained, and destained. In some systems, shrinking occurs during this operation and it is advisable to punch the lateral strips and the remaining gel slab with some guide marks as suggested by Graesslin et al. (15). Protein bands can be localized and cut out from the unstained slab. Proteins are eluted from the gel, as previously described (16), by grinding in an appropriate elution buffer followed by centrifugation and filtration through a 0.45~pm pore size Millipore filter. This procedure is repeated three to five times and the extracts are pooled and concentrated. Analytical procedure. Analytical gel slab electrophoresis is performed in a scaled-down manner with 1.5-mm-thick spacers and 15-cm-wide, 10 cm high glass plates. The procedure is essentially the same as the preparative one except in step v. Here, several small sample compart-
SIMPLIFIED
SLAB GEL ELECTROPHORESIS
357
FIG. 2. Photograph of preparative gel slab electrophoresis assembly. Four gel slabs in one bag are electrophoresed simultaneously. A centrifuge pump ensuring buffer circulation from the tank to the plastic bag is attached. The bag is pierced to allow return of the buffer to the lower chamber.
ments have to be formed instead of layering the sample directly on the gel surface. This is achieved by placing a well-forming Lucite comb (3,12,13) in the gel before polymerization or by inserting small separators like supple tubing (5). Up to 20 samples of 1 to 50 ~1 can be applied per S-cm gel slab and run as in the preparative method. Two slabs can be easily handled in one analytical run. RESULTS AND DISCUSSION
The procedure reported here is routinely used in our laboratory in place of the less simple apparatus of Kaltschmidt and Wittmann (10) for the isolation and purification of rat a-fetoprotein (AFP) variants (16), rat chloroleukemia H2B histone (17), rat monoclonal immunoglobulin (Rousseau, J., unpublished results), and myosin light chains (Tetaert, D., unpublished results). The unique feature of the procedure, providing simplicity and originality, is the use of a plastic bag in the two crucial steps: (i) polymerization of the gel slab and (ii) electrophoresis.
358
AAAAAAA A
J.P.KERCKAERT
SIMPLIFIED
SLAB
GEL
ELECTROPHORESIS
359
Applications of this method with two different gel systems will now be described. Serum proteins and many acidic substances can be separated in the high pH and discontinuous gel system of Davis (18). Figure 3A shows an entirely stained preparative slab of rat fetal serum proteins. Among about 20 bands, albumin (k), two AFP variants (i, j), and transferrins (e, f) account for the majority of the fetal serum proteins. From a similar but unstained slab, 11 different zones were excised, the proteins were then eluted in 10 mM phosphate buffer, pH 7.2-0.15 M sodium chloride, concentrated to 2 ml by ultrafiltration on a PM 30 Diaflo membrane, and subjected (10 ~1) to analytical electrophoresis (Fig. 3B). Generally after four extractions, 50-60% of the albumin or AFP is recovered in a pure form from 12% acrylamide gels, the concentration of which provides the best resolution of these proteins. In this highly concentrated gel, the slowest components of the serum (fractions a-h) are more difficult to excise from the slab without contamination, but more appropriate gel conditions can be found to overcome this problem. Basic proteins can be resolved on polyacrylamide gel at low pH. The method of Panyim and Chalkley (19) using an acetic acid buffer, pH 2.7, with a 17% acrylamide-0.113% bisacrylamide gel in 2.5 M urea provides good separation of histones as shown in Fig. 3C. Ten milligrams of 0.25 N HCl-soluble proteins from rat liver chromatin, dissolved in 2 ml of 0.01 N acetic acid-8 M urea-5% P-mercaptoethanol, were subjected to preparative electrophoresis under 120 V during 15 hr. The five main components in the order of increasing migration rate are Hl , H3, H2B, I-I2A, and H4 histones. Each corresponding migration zone was excised, extracted with 0.01 N HCl, and subjected to analytical gel electrophoresis (Fig. 3D). HI, H3, and H4 histones are easily obtained as single bands while H2B and H2A fractions show some overlap. As for serum proteins, the recovery of histones was 50-600/o. The technique described herein offers the following advantages: (i) Assembly of the apparatus and preparation of the gel is greatly simplified, very rapid, and virtually error proof, thus reducing the amount of time necessary for an experiment. (ii) It is easy to scale the method for analytical or preparative work with no increase in equipment needs or complexity. (iii) Multiple slabs can be formed and run simultaneously, affording the opportunity to analyze 50 or more samples at the same time. (iv) Because the plastic sack also serves as the upper buffer reservoir, the final assembly of the electrophoresis tank(s) is also simplified and more rapid (v). The cost of all materials necessary to perform this slab electrophoresis is very minimal and no machine shop-type fabrication is required.
360
J. P. KERCKAERT
ACKNOWLEDGMENTS This work was partly .supported by grants from the Institut National de la Sante et de la Recherche Medicale. The author wishes to thank Mrs. S. Quief for expert technical assistance, Professor J. Johnson for helpful discussion and preparation of the manuscript, and Professor G. Biserte for encouragement in this work.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16.
Raymond, S. (1962) Clin. C/tern. 8, 455-470. Narayan, K. A., Vogel, M., and Lawrence, J. M. (1965) Anal. Biochem. 12, 526-541. Tichy, H. (1966) Anal. Biochem. 17, 320-326. Woodworth, R. C., and Clark, L. G. (1967) Anal. B&hem. 18, 295-304. Akroyd, P. (1967) Anal. Biochem. 19, 399-410. Stuyvesant, W. V. (1967) Nature (London) 214, 405-407. Reid, M. S., and Bieleski, R. L. (1968) Anal. Biochem. 22, 374-381. Margolis, J., and Kenrick, K. G. (1%8) Anal. Biochem. 25, 347-362. Blattler, D. P. (1%9) Anal. Biochem. 27, 73-76. Kaltschmidt, E., and Wittmann, H. G. (1970) Anal. Biochem. 36, 401-412. Maurer, H. R., and Dati, F. A. (1972) Anal. Biochem. 46, 19-32. Roberts, R. M., and Jones, J. S. (1972) Anal. Biochem. 49, 592-597. Studier, F. W. (1973) J. Mol. Biol. 79, 237-248. Williams, J. A., Brand, J. M., and Bosman, T. (1973)Anal. Biochem. 51, 383-389. Graesslin, D., Weise, H. C., and Rick, M. (1976) Anal. Biochem. 71, 492-499. Kerckaert, J. P., Bayard, B., Quiet S., and Biserte, G. (1975) FEBS Lett. 53, 234-236.
17. Martinage, A., Sautibre, P., Kerckaert, J. P., and Biserte, G. (1976) Biochem. Biophys. Acta 420, 37-41. 18. Davis, B. J. (1%4)Ann. N. Y. Acad. Sci. 121, 404-427. 19. Panyim, S., and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346.
BIOCHEMISTRY
Highly
84,
354-360 (1978)
Simplified Analytical or Preparative Gel Electrophoresis
Slab
J. P. KERCKAERT Institut de Recherches sur le Cancer de Liile (Institut Jules Driessens) et Uniti no 124 INSERM, BP 3567. 59020 Lille Ct5de.x.France Received November 24, 1976; accepted August 29, 1977 A simple, rapid, and efficient procedure has been developed for performing analytical or preparative slab gel electrophoresis using only common laboratory materials which can be assembled without special tools or equipment. From one to four gel slabs of variable size can be made between glass plates embedded inside a watertight, supple plastic bag which is then used as the upper electrode buffer chamber. This technique has been applied, with different electrophoretic systems, for both the analysis and isolation of serum proteins and rat liver histones.
During the past 10 years a number of apparatus have been designed for electrophoresis on polyacrylamide gel slabs (l- 14). However, for many workers the construction or the use of such devices may present some difficulties, especially in obtaining a liquid-tight gel mold. This often requires precise machining or the use of sealing material, grease (l-5,7,8), melted agar (7,13), rubber gaskets (6,10-12), adhesive tape (2,9,14), or polyacrylamide plugs (10). Further, in connecting the gel with the electrode buffer, one often needs to install a filter paper bridge (5) or to mount and clamp tightly the gel slab on the electrophoresis apparatus (l-4,6-8,11,13,14). To eliminate all these operations, I have developed a very simple, inexpensive device which can be set up rapidly and which gives excellent results for both analytical and preparative experiments. Simplification is obtained by using a plastic bag, first to maintain the acrylamide solution in the polymerization cell and, second, to serve as the upper electrode chamber. In contrast to other simple electrophoretic devices, the procedure described here does not require the machining of special equipment. Only common materials are needed: glass plates, glass spacers, a plastic bag, rubber bands, clothes pins, a beaker, and a power supply with platinium electrodes. In addition, the flexibility of this procedure allows the use of various sizes of gel slabs with up to four slabs per experiment. 0003-2697178/0842-0354$02.00/O Copyright All rights
Q 1978 by Academic Press, Inc. of reproduction in any form reserved.
354
SIMPLIFIED
SLAB
GEL
355
ELECTROPHORESIS
METHODS Preparative procedure. The operating procedure is schematically divided into six steps as illustrated in Fig. 1. (i) To form the gel chamber, two glass plates (A) and two glass spacers (B) are placed together without any sealing device in a plastic bag (C). The spacers can be easily held in place by ensuring a gentle pressure on the glass plates with one’s fingers. The height of the bag is about two times that of the glass plates. Plastic bags of appropriate sizes can be made with the aid of a plastic sealer (Soud Sac, Calor, France) from bulk plastic sheets commonly used for book covering. The bag must be transparent, supple but tear resistant, watertight, and clean. Generally, 20 x 20-cm plates with 0.5-cm-thick glass spacers (0.5 x 1.5 x 20 cm) are used. Up to four gel slabs can be polymerized simultaneously using five glass plates separated by four pairs of spacers. (ii) The bag is folded if too large and is pressed against the gel chamber by two outside glass plates (D). The assembly is secured with
El
El
H-
FIG. 1. Schematic representation of preparative slab gel electrophoresis. Glass plates; (B) glass spacers; (C) supple watertight plastic bag; (E) rubber acrylamide solution; (G) isobutanol overlay: (H) cutting off the bottom of the bag; to elevate the assembly; (K) sample syringe; (L) electrode buffer; (M) platinium Explanations of the different steps are detailed under Methods.
(A and D) bands; (F) (J) wedges electrodes.
356
J. P. KERCKAERT
rubber bands (E). Plates and spacers can then be readjusted, if necessary, inside the bag. (iii) The gel monomer solution (F) is poured into the bag to about 5 cm from the top of the plates. The gel is then overlaid with the aid of a Pasteur pipet with water-saturated isobutyl alcohol (G) to produce a flat surface and the gel is allowed to polymerize. (iv) After polymerization the surface of the gel is rinsed with distilled water and the bottom of the bag is cut off(H). (v) The assembly is placed vertically on two small (0.5 cm thick) glass or acrylic wedges (J) in a beaker or other tank. The surface of the gel must be horizontal to avoid skewed bands. The tank is used as the lower electrode chamber and it must be high enough to allow for attachment of the plastic bag to the walls of the chamber with clothes pins or paper clips. Electrode buffer is then poured in the tank and in the bag until the levels in these two compartments are the same. This eliminates pressure on the plastic sack and gives it a more suitable shape and a larger volume. Samples (1 to 5 ml per slab, containing 10 to 100 mg of proteins) made denser with 20% (w/v) glycerol or sucrose or with 8 M urea, are layered on the gel surface with the aid of a syringe fitted with a short piece of tubing (K). A trace of bromophenol blue (for alkaline gels) or pyronin red (for acidic gels) is added to the sample to visualize the layering. (vi) Platinium electrodes (M) are placed in the two buffer chambers and connected to a power supply. Up to 100 mA/slab with 80-150 Volts can be applied without excessive heating of the gel. Generally electrophoresis is carried out overnight and, if necessary, to avoid changes in buffer composition, an aquarium centrifuge pump can be installed as illustrated in Fig. 2 to allow buffer circulation from the lower to the upper electrode chamber. After completion of electrophoresis, the bag and the glass plates are disassembled. Two lateral guide strips of gel (1 cm large) are cut out, stained, and destained. In some systems, shrinking occurs during this operation and it is advisable to punch the lateral strips and the remaining gel slab with some guide marks as suggested by Graesslin et al. (15). Protein bands can be localized and cut out from the unstained slab. Proteins are eluted from the gel, as previously described (16), by grinding in an appropriate elution buffer followed by centrifugation and filtration through a 0.45~pm pore size Millipore filter. This procedure is repeated three to five times and the extracts are pooled and concentrated. Analytical procedure. Analytical gel slab electrophoresis is performed in a scaled-down manner with 1.5-mm-thick spacers and 15-cm-wide, 10 cm high glass plates. The procedure is essentially the same as the preparative one except in step v. Here, several small sample compart-
SIMPLIFIED
SLAB GEL ELECTROPHORESIS
357
FIG. 2. Photograph of preparative gel slab electrophoresis assembly. Four gel slabs in one bag are electrophoresed simultaneously. A centrifuge pump ensuring buffer circulation from the tank to the plastic bag is attached. The bag is pierced to allow return of the buffer to the lower chamber.
ments have to be formed instead of layering the sample directly on the gel surface. This is achieved by placing a well-forming Lucite comb (3,12,13) in the gel before polymerization or by inserting small separators like supple tubing (5). Up to 20 samples of 1 to 50 ~1 can be applied per S-cm gel slab and run as in the preparative method. Two slabs can be easily handled in one analytical run. RESULTS AND DISCUSSION
The procedure reported here is routinely used in our laboratory in place of the less simple apparatus of Kaltschmidt and Wittmann (10) for the isolation and purification of rat a-fetoprotein (AFP) variants (16), rat chloroleukemia H2B histone (17), rat monoclonal immunoglobulin (Rousseau, J., unpublished results), and myosin light chains (Tetaert, D., unpublished results). The unique feature of the procedure, providing simplicity and originality, is the use of a plastic bag in the two crucial steps: (i) polymerization of the gel slab and (ii) electrophoresis.
358
AAAAAAA A
J.P.KERCKAERT
SIMPLIFIED
SLAB
GEL
ELECTROPHORESIS
359
Applications of this method with two different gel systems will now be described. Serum proteins and many acidic substances can be separated in the high pH and discontinuous gel system of Davis (18). Figure 3A shows an entirely stained preparative slab of rat fetal serum proteins. Among about 20 bands, albumin (k), two AFP variants (i, j), and transferrins (e, f) account for the majority of the fetal serum proteins. From a similar but unstained slab, 11 different zones were excised, the proteins were then eluted in 10 mM phosphate buffer, pH 7.2-0.15 M sodium chloride, concentrated to 2 ml by ultrafiltration on a PM 30 Diaflo membrane, and subjected (10 ~1) to analytical electrophoresis (Fig. 3B). Generally after four extractions, 50-60% of the albumin or AFP is recovered in a pure form from 12% acrylamide gels, the concentration of which provides the best resolution of these proteins. In this highly concentrated gel, the slowest components of the serum (fractions a-h) are more difficult to excise from the slab without contamination, but more appropriate gel conditions can be found to overcome this problem. Basic proteins can be resolved on polyacrylamide gel at low pH. The method of Panyim and Chalkley (19) using an acetic acid buffer, pH 2.7, with a 17% acrylamide-0.113% bisacrylamide gel in 2.5 M urea provides good separation of histones as shown in Fig. 3C. Ten milligrams of 0.25 N HCl-soluble proteins from rat liver chromatin, dissolved in 2 ml of 0.01 N acetic acid-8 M urea-5% P-mercaptoethanol, were subjected to preparative electrophoresis under 120 V during 15 hr. The five main components in the order of increasing migration rate are Hl , H3, H2B, I-I2A, and H4 histones. Each corresponding migration zone was excised, extracted with 0.01 N HCl, and subjected to analytical gel electrophoresis (Fig. 3D). HI, H3, and H4 histones are easily obtained as single bands while H2B and H2A fractions show some overlap. As for serum proteins, the recovery of histones was 50-600/o. The technique described herein offers the following advantages: (i) Assembly of the apparatus and preparation of the gel is greatly simplified, very rapid, and virtually error proof, thus reducing the amount of time necessary for an experiment. (ii) It is easy to scale the method for analytical or preparative work with no increase in equipment needs or complexity. (iii) Multiple slabs can be formed and run simultaneously, affording the opportunity to analyze 50 or more samples at the same time. (iv) Because the plastic sack also serves as the upper buffer reservoir, the final assembly of the electrophoresis tank(s) is also simplified and more rapid (v). The cost of all materials necessary to perform this slab electrophoresis is very minimal and no machine shop-type fabrication is required.
360
J. P. KERCKAERT
ACKNOWLEDGMENTS This work was partly .supported by grants from the Institut National de la Sante et de la Recherche Medicale. The author wishes to thank Mrs. S. Quief for expert technical assistance, Professor J. Johnson for helpful discussion and preparation of the manuscript, and Professor G. Biserte for encouragement in this work.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16.
Raymond, S. (1962) Clin. C/tern. 8, 455-470. Narayan, K. A., Vogel, M., and Lawrence, J. M. (1965) Anal. Biochem. 12, 526-541. Tichy, H. (1966) Anal. Biochem. 17, 320-326. Woodworth, R. C., and Clark, L. G. (1967) Anal. B&hem. 18, 295-304. Akroyd, P. (1967) Anal. Biochem. 19, 399-410. Stuyvesant, W. V. (1967) Nature (London) 214, 405-407. Reid, M. S., and Bieleski, R. L. (1968) Anal. Biochem. 22, 374-381. Margolis, J., and Kenrick, K. G. (1%8) Anal. Biochem. 25, 347-362. Blattler, D. P. (1%9) Anal. Biochem. 27, 73-76. Kaltschmidt, E., and Wittmann, H. G. (1970) Anal. Biochem. 36, 401-412. Maurer, H. R., and Dati, F. A. (1972) Anal. Biochem. 46, 19-32. Roberts, R. M., and Jones, J. S. (1972) Anal. Biochem. 49, 592-597. Studier, F. W. (1973) J. Mol. Biol. 79, 237-248. Williams, J. A., Brand, J. M., and Bosman, T. (1973)Anal. Biochem. 51, 383-389. Graesslin, D., Weise, H. C., and Rick, M. (1976) Anal. Biochem. 71, 492-499. Kerckaert, J. P., Bayard, B., Quiet S., and Biserte, G. (1975) FEBS Lett. 53, 234-236.
17. Martinage, A., Sautibre, P., Kerckaert, J. P., and Biserte, G. (1976) Biochem. Biophys. Acta 420, 37-41. 18. Davis, B. J. (1%4)Ann. N. Y. Acad. Sci. 121, 404-427. 19. Panyim, S., and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346.
