ANALYTICAL
BIOCHEMISTRY
87, 287-292 (1978)
Two-Dimensional Electrophoresis: An Application Combining Cellulose Acetate Membrane with SDS-Polyacrylamide Gradient Gel A simple, effective method for high-resolution two-dimensional electrophoresis is described. The combination of cellulose acetate with SDS-polyacrylamide gradient gels has been studied for a range of standard proteins and for comparison of lupine seed storage proteins. The speed of the electrophoretic separation and the general ease of handling of cellulose acetate membranes make them ideal for the first dimension. Furthermore, because these membranes are very thin, reduction with P-mercaptoethanol and incubation with SDS can be achieved immediately on top of the second-dimension slab.
Two-dimensional mapping techniques, employing different kinds of electrophoresis in polyacrylamide gel, are becoming increasingly popular for the resolution of complex protein mixtures (l-4). In most cases the aim has been to resolve as many polypeptides as possible with dissociating conditions being used in each dimension. While this is satisfactory for proteins which contain identical subunits, there may be difficulties in interpretation if the proteins contain different numbers of nonidentical subunits. In such cases, there is often the need simply to separate the proteins into groups under nondissociating conditions in the first dimension. A high-resolution electrophoretic procedure employing denaturing conditions can then be used for the second dimension in order to compare the proteins in these groups. It has been suggested (5) that electrophoresis on cellulose acetate followed by isoelectric focusing or electrophoresis on step-gradient polyacrylamide gel could be used for the separation of serum proteins. These authors covered the cellulose acetate membrane with rapidly polymerizing polyacrylamide gel solution to prevent the components on the strip from diffusing and washing into each other. In another report (6), cellulose acetate strips were embedded into a normal polyacrylamide gel for the separation of ribosomal proteins. We have found that this is not necessary; we have successfully separated proteins on cellulose acetate and transferred them directly into sodium dodecyl sulphate (SDS)-polyacrylamide gradient gels. In this report we describe results which show that proteins can be reduced with /3-mercaptoethanol and dissociated with SDS without separate incubation of the cellulose acetate or addition of polymerizing solution. The application of this technique to the separation of seed storage globulins is demonstrated by comparison of the proteins from two species of lupine. 287
0003-2697/78/0871-0287$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
288
SHORT COMMUNICATIONS
METHODS
Polyacrylamide gradient gels (Gradipore, 2.5-27% concave gradient, variable crosslinkage, from Gradient Pty. Ltd., Lane Cove, Australia) were used for the second dimension. These gels were subjected to preelectrophoresis in 0.025 M phosphate buffer, 0.15% (w/v) SDS for 2 hr at 50 mA in a Gradipore single-cell electrophoresis unit. If reduction of disulfide bonds was required, preelectrophoresis was followed by the addition of 0.25 ml of P-mercaptoethanol, which was pipetted onto the gel and allowed to stand for at least 30 min. Complete reduction did not occur if the gel was not treated in this manner, while attempts to use alternative reducing agents (thioglycolic acid and dithiothreitol) were not as successful. Before applying the strip of cellulose acetate, excess P-mercaptoethanol was drained away, and the glass containing the slab was carefully blotted dry to facilitate handling of the membrane. Cellulose acetate electrophoresis was performed using a Beckman Microzone electrophoresis system. Besides the samples loaded for twodimensional electrophoresis, a separate set was loaded for staining and photography. The portion of the membrane containing the separated proteins for the second dimension was carefully excised and gently laid on top of the polyacrylamide slab without trapping bubbles. After covering with SDS-phosphate buffer, second-dimensional electrophoresis (50 mA, approx, 70 V) was commenced immediately. Samples containing the original
FIG. 1. Two-dimensional cellulose acetate SDS-polyacrylamide gradient gel electrophoresis of standard proteins in phosphate-SDS buffer, pH 7. The first dimension was run from left to right, 150 V for 20 min in 0.05 M phosphate buffer, pH 7. The proteins (from left to right) lysozyme, myoglobin, tropomyosin fumarase, and BSA were dissolved in 0.15 M phosphate buffer, pH 7, and approximately 0.5 gg of each Ioaded (excised portion of membrane contained approx. 0.2 pg). The second dimension was run from top to bottom. A mixture of the proteins was incubated in 0.05 M phosphate buffer, 2% (w/v) SDS, pH 7, at 100°C for 10 min and loaded (approx. 0.5 pg of each) for comparison on the left-hand side.
SHORT
COMMUNICATIONS
289
mixture of proteins, incubated by heating with SDS at 100°C for 10 min, were quickly loaded at the end of the cellulose acetate strip. A single slot, cut from the plastic spacer provided with the Gradipore apparatus, was used for this purpose. The power was briefly switched off to allow loading of the samples, but not while the slot was being inserted. The slot spacer and the acetate strip were removed after 15 min, and electrophoresis was continued for an additional 3.5 hr. The gradient gels were stained with Coomassie brilliant blue G250 (ICI) in the normal manner (7). The remaining cellulose acetate membrane was stained with this dye by the procedure of Blagrove et al. (8). RESULTS
AND DISCUSSION
Many two-dimensional electrophoresis systems use SDS-polyacrylamide gel for the second dimension in order to study both the number and approximate molecular weight of the subunit polypeptides. Normally, if a polyacrylamide disk or slab gel has been used as a first dimension, this gel is soaked in buffer containing SDS in order to bind the detergent to the protein. It is generally assumed that this is sufficient to give proper incubation for the second dimension. In order to demonstrate that proteins separated by electrophoresis on cellulose acetate and simply laid on top of a gradient gel could be reduced with P-mercaptoethanol and bind SDS in the normal fashion, a number of standard proteins were studied. To achieve the separation shown in Fig. 1, lysozyme, myoglobin, and tropomyosin were mixed and loaded towards the cathode, whereas fumarase and BSA were applied together near the center of the cellulose acetate membrane. It can be seen in Fig. 1 that each of these proteins gave a band in the second dimension with the same relative mobility as that found for reaction with SDS under more vigorous conditions. A similar separation of standard proteins, on the basis of molecular size, has been reported for SDS-polyacrylamide gradient gel electrophoresis in one dimension (9). The similarity between the bands for the incubated mixture and samples loaded on cellulose acetate is remarkable. The tendency to be either sharp or diffuse is reproduced independently of the different conditions for interaction with SDS. It has been reported (10) that some proteins require vigorous treatment for complete dissociation in SDS solution, and presumably such samples would not be suited to the present technique. However, by inclusion of a properly incubated mixture for comparison, it should be possible to determine if complete reduction and binding has occurred. It has been found that spurious stained regions may appear on gels treated with p-mercaptoethanol. These can be readily discriminated from the protein bands by alignment with the cellulose acetate strip and comparison with the standard mixture. It has been shown that electrophoresis on cellulose acetate is a useful
290
SHORT COMMUNICATIONS
FIG. 2. Two-dimensional cellulose acetate SDS-polyacrylamide gradient gel electrophoreL.H.S. and L. pilosus R.H.S.: (A) without reduction; (B) after sis ofLupinus angustifolius reduction with P-mercaptoethanol. The first dimension was run from left to right, 250 V for 10 min in 0.05 M phosphate buffer, pH 7. The seed extracts (I I) were dissolved in 0.15 M phosphate buffer, pH 7, and 5 pg of L. angustifolius and 2.5 pg of L. pilosus were loaded near the cathode and center of strip, respectively (excised portion of membrane contained approx. halfofthis protein). The separated proteins, congiutins (Y,6, and y, are shown for each species. The second dimension was run from top to bottom. The whole-seed extract from each species was incubated in 0.05 M phosphate buffer, 2% (w/v) SDS, pH 7, at 100°C for 10 min and loaded (approx. 5 pg) for comparison on the left and right side, respectively.
technique for the study of legume seed proteins (11). In particular, lupine seed extracts can be resolved into three protein bands, conglutins CX,p, and y, in only 10 min. Tedious chemical fractionation was necessary in order to compare the subunit patterns for these proteins on SDS-polyacrylamide gel (11). While two-dimensional electrophoresis using acidic urea-poly-
SHORT
COMMUNICATIONS
291
acrylamide followed by SDS-polyacrylamide gel gives good resolution of these proteins into a large number of subunit polypeptides, it does not indicate from which protein fraction each polypeptide was derived. However, two-dimensional cellulose acetate/SDS-polyacrylamide gradient gel electrophoresis allows direct determination of the subunit pattern for each of the lupine globulins before and after reduction of disulfide bonds (Fig. 2). Furthermore, two or more seed extracts can be loaded at different positions on the membrane and compared directly on the same gradient gel. For example, Fig. 2 compares two lupine species which have similar cellulose acetate electrophoretic patterns. Two major features are worthy of comment. First, most of the bands seen in the complex subunit pattern for the whole-seed extract can be related to the results seen for the separate proteins. Secondly, there are differences between these species in the subunit pattern for conglutins (Yand p but not for conglutin y. Reduction with P-mercaptoethanol shows further differences between the conglutin (Yfractions, whereas conglutin /3 is not affected. Although the proportion of conglutin y is low in these species, faint bands corresponding to the reduced form (11) of this protein were seen for both species before photography. Not only does this technique greatly facilitate comparison between proteins from different lupine genotypes, it also allows the study of the effect of environment on the proportion and kind of polypeptides synthesized. The limited use of cellulose acetate for two-dimensional electrophoresis stems from its lack of resolution when compared with other gel techniques. However, the advantages of speed and ease of handling make it an ideal first dimension for proteins which can be separated on the basis of charge differences rather than molecular sieving. Besides the method reported here, we have separated proteins on cellulose acetate and electrophoresed them directly into an acidic urea-polyacrylamide gel slab. Because a sample applied in a cellulose acetate membrane is in a very thin layer, the second dimension need not specifically sharpen the electrophoretic zones. This contrasts with sample application from polyacrylamide disks or slabs which generally requires a procedure whereby the zones are sharpened in order to give satisfactory results. We conclude that cellulose acetate should be considered as a medium for two-dimensional electrophoresis since it has advantages in applications such as those reported above. By using commercially available polyacrylamide gradient gels in combination with cellulose acetate, large numbers of separations can be made quickly and easily with minimal laboratory assistance. The ease of the method is further enhanced by the design of the cellulose acetate electrophoresis system which allows accurate application of samples at different positions along the membrane.
292
SHORT COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Dale, G., and Lamer, A. L. (1969) C/in. Chim. Acta 24, 61-68. Kenrick, K. G., and Margolis, I. (1970) Anal. Biochem. 33, 204-207. Kahschmidt, E., and Wittmann, H. G. (1970) Anal. Biochem. 36, 401-412. O’Farrell, P. H. (1975)J. Biol. Chem. 250, 4007-4021. Maurer, H. R., and Allen, R. C. (1972) C/in. Chim. Acta 40, 359-370. Lin, A., Collatz, E., and Wool, I. G. (1976) Mol. Gen. Gene!. 144, l-9. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412. Blagrove. R. J.. Frenkel, M. J., and Gillespie, J. M. (1975) Comp. Biochem. 5OB, 571-572. 9. Lambin, P., Rochu, D., and Fine, J. M. (1976) Anal. Biochem. 74, 567-575. 10. Deutsch, D. G. (1976) Anal. Biochem. 71, 300-303. Il. Blagrove, R. J., and Gillespie, J. M. (1975) Aust. J. P/ant Physiol. 2, 13-27.
Physiol.
R. J. BLAGROVE M. J. FRENKEL Division of Protein Chemistry, CSIRO 343 Royal Parade Parkville, Victoria 3052. Australia Received November 28, 1977; accepted
January
27, 1978
BIOCHEMISTRY
87, 287-292 (1978)
Two-Dimensional Electrophoresis: An Application Combining Cellulose Acetate Membrane with SDS-Polyacrylamide Gradient Gel A simple, effective method for high-resolution two-dimensional electrophoresis is described. The combination of cellulose acetate with SDS-polyacrylamide gradient gels has been studied for a range of standard proteins and for comparison of lupine seed storage proteins. The speed of the electrophoretic separation and the general ease of handling of cellulose acetate membranes make them ideal for the first dimension. Furthermore, because these membranes are very thin, reduction with P-mercaptoethanol and incubation with SDS can be achieved immediately on top of the second-dimension slab.
Two-dimensional mapping techniques, employing different kinds of electrophoresis in polyacrylamide gel, are becoming increasingly popular for the resolution of complex protein mixtures (l-4). In most cases the aim has been to resolve as many polypeptides as possible with dissociating conditions being used in each dimension. While this is satisfactory for proteins which contain identical subunits, there may be difficulties in interpretation if the proteins contain different numbers of nonidentical subunits. In such cases, there is often the need simply to separate the proteins into groups under nondissociating conditions in the first dimension. A high-resolution electrophoretic procedure employing denaturing conditions can then be used for the second dimension in order to compare the proteins in these groups. It has been suggested (5) that electrophoresis on cellulose acetate followed by isoelectric focusing or electrophoresis on step-gradient polyacrylamide gel could be used for the separation of serum proteins. These authors covered the cellulose acetate membrane with rapidly polymerizing polyacrylamide gel solution to prevent the components on the strip from diffusing and washing into each other. In another report (6), cellulose acetate strips were embedded into a normal polyacrylamide gel for the separation of ribosomal proteins. We have found that this is not necessary; we have successfully separated proteins on cellulose acetate and transferred them directly into sodium dodecyl sulphate (SDS)-polyacrylamide gradient gels. In this report we describe results which show that proteins can be reduced with /3-mercaptoethanol and dissociated with SDS without separate incubation of the cellulose acetate or addition of polymerizing solution. The application of this technique to the separation of seed storage globulins is demonstrated by comparison of the proteins from two species of lupine. 287
0003-2697/78/0871-0287$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
288
SHORT COMMUNICATIONS
METHODS
Polyacrylamide gradient gels (Gradipore, 2.5-27% concave gradient, variable crosslinkage, from Gradient Pty. Ltd., Lane Cove, Australia) were used for the second dimension. These gels were subjected to preelectrophoresis in 0.025 M phosphate buffer, 0.15% (w/v) SDS for 2 hr at 50 mA in a Gradipore single-cell electrophoresis unit. If reduction of disulfide bonds was required, preelectrophoresis was followed by the addition of 0.25 ml of P-mercaptoethanol, which was pipetted onto the gel and allowed to stand for at least 30 min. Complete reduction did not occur if the gel was not treated in this manner, while attempts to use alternative reducing agents (thioglycolic acid and dithiothreitol) were not as successful. Before applying the strip of cellulose acetate, excess P-mercaptoethanol was drained away, and the glass containing the slab was carefully blotted dry to facilitate handling of the membrane. Cellulose acetate electrophoresis was performed using a Beckman Microzone electrophoresis system. Besides the samples loaded for twodimensional electrophoresis, a separate set was loaded for staining and photography. The portion of the membrane containing the separated proteins for the second dimension was carefully excised and gently laid on top of the polyacrylamide slab without trapping bubbles. After covering with SDS-phosphate buffer, second-dimensional electrophoresis (50 mA, approx, 70 V) was commenced immediately. Samples containing the original
FIG. 1. Two-dimensional cellulose acetate SDS-polyacrylamide gradient gel electrophoresis of standard proteins in phosphate-SDS buffer, pH 7. The first dimension was run from left to right, 150 V for 20 min in 0.05 M phosphate buffer, pH 7. The proteins (from left to right) lysozyme, myoglobin, tropomyosin fumarase, and BSA were dissolved in 0.15 M phosphate buffer, pH 7, and approximately 0.5 gg of each Ioaded (excised portion of membrane contained approx. 0.2 pg). The second dimension was run from top to bottom. A mixture of the proteins was incubated in 0.05 M phosphate buffer, 2% (w/v) SDS, pH 7, at 100°C for 10 min and loaded (approx. 0.5 pg of each) for comparison on the left-hand side.
SHORT
COMMUNICATIONS
289
mixture of proteins, incubated by heating with SDS at 100°C for 10 min, were quickly loaded at the end of the cellulose acetate strip. A single slot, cut from the plastic spacer provided with the Gradipore apparatus, was used for this purpose. The power was briefly switched off to allow loading of the samples, but not while the slot was being inserted. The slot spacer and the acetate strip were removed after 15 min, and electrophoresis was continued for an additional 3.5 hr. The gradient gels were stained with Coomassie brilliant blue G250 (ICI) in the normal manner (7). The remaining cellulose acetate membrane was stained with this dye by the procedure of Blagrove et al. (8). RESULTS
AND DISCUSSION
Many two-dimensional electrophoresis systems use SDS-polyacrylamide gel for the second dimension in order to study both the number and approximate molecular weight of the subunit polypeptides. Normally, if a polyacrylamide disk or slab gel has been used as a first dimension, this gel is soaked in buffer containing SDS in order to bind the detergent to the protein. It is generally assumed that this is sufficient to give proper incubation for the second dimension. In order to demonstrate that proteins separated by electrophoresis on cellulose acetate and simply laid on top of a gradient gel could be reduced with P-mercaptoethanol and bind SDS in the normal fashion, a number of standard proteins were studied. To achieve the separation shown in Fig. 1, lysozyme, myoglobin, and tropomyosin were mixed and loaded towards the cathode, whereas fumarase and BSA were applied together near the center of the cellulose acetate membrane. It can be seen in Fig. 1 that each of these proteins gave a band in the second dimension with the same relative mobility as that found for reaction with SDS under more vigorous conditions. A similar separation of standard proteins, on the basis of molecular size, has been reported for SDS-polyacrylamide gradient gel electrophoresis in one dimension (9). The similarity between the bands for the incubated mixture and samples loaded on cellulose acetate is remarkable. The tendency to be either sharp or diffuse is reproduced independently of the different conditions for interaction with SDS. It has been reported (10) that some proteins require vigorous treatment for complete dissociation in SDS solution, and presumably such samples would not be suited to the present technique. However, by inclusion of a properly incubated mixture for comparison, it should be possible to determine if complete reduction and binding has occurred. It has been found that spurious stained regions may appear on gels treated with p-mercaptoethanol. These can be readily discriminated from the protein bands by alignment with the cellulose acetate strip and comparison with the standard mixture. It has been shown that electrophoresis on cellulose acetate is a useful
290
SHORT COMMUNICATIONS
FIG. 2. Two-dimensional cellulose acetate SDS-polyacrylamide gradient gel electrophoreL.H.S. and L. pilosus R.H.S.: (A) without reduction; (B) after sis ofLupinus angustifolius reduction with P-mercaptoethanol. The first dimension was run from left to right, 250 V for 10 min in 0.05 M phosphate buffer, pH 7. The seed extracts (I I) were dissolved in 0.15 M phosphate buffer, pH 7, and 5 pg of L. angustifolius and 2.5 pg of L. pilosus were loaded near the cathode and center of strip, respectively (excised portion of membrane contained approx. halfofthis protein). The separated proteins, congiutins (Y,6, and y, are shown for each species. The second dimension was run from top to bottom. The whole-seed extract from each species was incubated in 0.05 M phosphate buffer, 2% (w/v) SDS, pH 7, at 100°C for 10 min and loaded (approx. 5 pg) for comparison on the left and right side, respectively.
technique for the study of legume seed proteins (11). In particular, lupine seed extracts can be resolved into three protein bands, conglutins CX,p, and y, in only 10 min. Tedious chemical fractionation was necessary in order to compare the subunit patterns for these proteins on SDS-polyacrylamide gel (11). While two-dimensional electrophoresis using acidic urea-poly-
SHORT
COMMUNICATIONS
291
acrylamide followed by SDS-polyacrylamide gel gives good resolution of these proteins into a large number of subunit polypeptides, it does not indicate from which protein fraction each polypeptide was derived. However, two-dimensional cellulose acetate/SDS-polyacrylamide gradient gel electrophoresis allows direct determination of the subunit pattern for each of the lupine globulins before and after reduction of disulfide bonds (Fig. 2). Furthermore, two or more seed extracts can be loaded at different positions on the membrane and compared directly on the same gradient gel. For example, Fig. 2 compares two lupine species which have similar cellulose acetate electrophoretic patterns. Two major features are worthy of comment. First, most of the bands seen in the complex subunit pattern for the whole-seed extract can be related to the results seen for the separate proteins. Secondly, there are differences between these species in the subunit pattern for conglutins (Yand p but not for conglutin y. Reduction with P-mercaptoethanol shows further differences between the conglutin (Yfractions, whereas conglutin /3 is not affected. Although the proportion of conglutin y is low in these species, faint bands corresponding to the reduced form (11) of this protein were seen for both species before photography. Not only does this technique greatly facilitate comparison between proteins from different lupine genotypes, it also allows the study of the effect of environment on the proportion and kind of polypeptides synthesized. The limited use of cellulose acetate for two-dimensional electrophoresis stems from its lack of resolution when compared with other gel techniques. However, the advantages of speed and ease of handling make it an ideal first dimension for proteins which can be separated on the basis of charge differences rather than molecular sieving. Besides the method reported here, we have separated proteins on cellulose acetate and electrophoresed them directly into an acidic urea-polyacrylamide gel slab. Because a sample applied in a cellulose acetate membrane is in a very thin layer, the second dimension need not specifically sharpen the electrophoretic zones. This contrasts with sample application from polyacrylamide disks or slabs which generally requires a procedure whereby the zones are sharpened in order to give satisfactory results. We conclude that cellulose acetate should be considered as a medium for two-dimensional electrophoresis since it has advantages in applications such as those reported above. By using commercially available polyacrylamide gradient gels in combination with cellulose acetate, large numbers of separations can be made quickly and easily with minimal laboratory assistance. The ease of the method is further enhanced by the design of the cellulose acetate electrophoresis system which allows accurate application of samples at different positions along the membrane.
292
SHORT COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Dale, G., and Lamer, A. L. (1969) C/in. Chim. Acta 24, 61-68. Kenrick, K. G., and Margolis, I. (1970) Anal. Biochem. 33, 204-207. Kahschmidt, E., and Wittmann, H. G. (1970) Anal. Biochem. 36, 401-412. O’Farrell, P. H. (1975)J. Biol. Chem. 250, 4007-4021. Maurer, H. R., and Allen, R. C. (1972) C/in. Chim. Acta 40, 359-370. Lin, A., Collatz, E., and Wool, I. G. (1976) Mol. Gen. Gene!. 144, l-9. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412. Blagrove. R. J.. Frenkel, M. J., and Gillespie, J. M. (1975) Comp. Biochem. 5OB, 571-572. 9. Lambin, P., Rochu, D., and Fine, J. M. (1976) Anal. Biochem. 74, 567-575. 10. Deutsch, D. G. (1976) Anal. Biochem. 71, 300-303. Il. Blagrove, R. J., and Gillespie, J. M. (1975) Aust. J. P/ant Physiol. 2, 13-27.
Physiol.
R. J. BLAGROVE M. J. FRENKEL Division of Protein Chemistry, CSIRO 343 Royal Parade Parkville, Victoria 3052. Australia Received November 28, 1977; accepted
January
27, 1978
