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Rapid isoelectric focusing in a vertical polyacrylamide minigel system
ANALYTICAL
BIOCHEMISTRY
Rapid Isoelectric
167,290-294
(1987)
Focusing in a Vertical Polyacrylamide
Minigel System
EUGENE F. ROBERTSON,H.KATHLEENDANNELLY,PETERJ.MALLOY, ANDHENRYC.REEVES Department of Botany and Microbiology, Arizona State University, Tempe. Arizona
85287
Received April 24, 1987 A rapid method is described for the resolution of proteins employing isoelectric focusing in a vertical polyacrylamide minigel system. Isoelectric focusing can be performed in only 3 h, utilizing low voltage, under either native conditions or denaturing conditions in the presence of 8 M urea. The procedure permits the application of larger sample volumes containing more protein than other isoelectric focusing procedures, and provides the additional advantages of slab gels over tube gels for analytical purposes. The procedure is also well adapted for use in two-dimensional electrophoretic techniques, making it possible to complete a two-dimensional gel in 1 day. 0 1987 Academic Press, Inc. KEY WORDS: isoelectric focusing; electrophoresis; two-dimensional electrophoresis; protein; polyacrylamide gel; minigel.
Isoelectric focusing is in itself a powerful technique for the resolution of complex protein mixtures and when used under denaturing conditions as part of a two-dimensional analysis as many as 1,100 proteins can be resolved (1). This paper introduces a novel procedure for isoelectric focusing in vertical polyacrylamide minigels with several advantages over other methods that serve the same purpose. Isoelectric focusing in tube gels presents difficulties in the analysis of results due to the cylindrical configuration of the gel and problems often associated with comparing protein patterns in multiple cast gels. These problems can be avoided by employing horizontal slab gel isoelectric focusing; however, this procedure is limited by the inability to apply large sample volumes containing high concentrations of protein and the necessity for an efficient cooling plate on which to place the gel during the run (2). Isoelectric focusing performed in standard vertical, rather than horizontal, gels allows for the application of larger sample volumes but requires either long running times or high voltages (3,4). The method described in this paper eliminates these often encoun0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
tered problems and combines the advantages of the three commonly used methods for isoelectric focusing. MATERIALS
AND METHODS
Materials. Electrophoresis grade acrylamide and bis-acrylamide, N,N,N’, N’-tetramethylenediamine (TEMED),’ and ammonium persulfate were obtained from Bio-Rad Laboratories. Coomassie brilliant blue R-250 and isoelectric focusing standards in the range 3.55 to 9.3 were from Sigma Chemical Co. Ampholytes, in the pH range 3 to 10 and 4 to 7, were from Serva Fine Biochemicals. Sequanal-grade urea was purchased from Pierce Chemical Co. Native IEF. A vertical minigel system with an 8 X 7-cm cell format was utilized. The gels were cast 1.5 mm thick from the following mixture: 7 ml water, 2 ml acrylamide mixture (30% (w/v) acrylamide, 1% (w/v) bis-acrylamide), 2.4 ml 50% (v/v) glycerol, ’ Abbreviations used: IEF, isoelectric focusing: SDS, sodium dodecyl sulfate, TEMED, N,N,N’,N’-tetramethylenediamine. 290
MINIGEL
ISOELECTRIC
291
FOCUSING
I 2 Migration
4 Distance
(cm)
6
FIG. 1. Protein standards were analyzed by isoelectric focusing in the pH range 3-10 as described in the text. The three lanes in (A) each contain 16 rg of Sigma IEF 3.55-9.3 standard protein mix, applied in 20 ~1. The pH gradient was measured with a flat bottom pH electrode on the gel surface, and is indicated along the side of (A). The gel was stained for protein with Coomassie blue. The reported isoelectric points of the standard proteins were plotted against their migration distance from the bottom of the sample wells, and the best fit line is shown in (B). Published pI values for standard proteins are as follows: amyloglucosidase, 3.55; trypsin inhibitor, 4.55; @-Iactoglobulin A, 5.13; bovine carbonic anhydrase, 5.85; human carbonic anhydrase, 6.57; myoglobin, 6.76,7.16*; lactic dehydrogenase, 8.3,8.4,8.55*; trypsinogen, 9.3*. (*Indicates not visible in (A)).
and 0.6 ml ampholyte (pH range 3-10). These components were mixed, degassed, and then 50 ~1 of 10% (w/v) ammonium persulfate and 20 ~1 TEMED were added. Gels were cast with a lo-well comb in place and allowed to polymerize for 1 h. The cathode solution was 25 mM NaOH and the anode solution 20 mM acetic acid. These solutions were cooled to 4°C prior to electrophoresis. After polymerization was complete, the comb was removed and the wells were rinsed and then filled with the cathode solution. Protein standards were
mixed with an equal volume of 60% (v/v) glycerol and 4% (v/v) ampholyte of the same pH range used to prepare the gel. Electrophoresis was performed at room temperature for 1.5 h at 200 V constant voltage, then increased to 400 V constant voltage for an additional 1.5 h. After electrophoresis was complete the gel was rinsed briefly with distilled water and the pH gradient was measured on the gel surface with a flat bottom pH electrode. Denaturing ZEF. Isoelectric focusing gels containing 8 M urea were cast in a vertical 8
292
ROBERTSON
X 7-cm cell format as above. The mixture for a 1.5-mm gel contained 2.7 ml water, 2.0 ml acrylamide mixture (see above), 2.4 ml 50% glycerol, 0.6 ml Serva ampholyte (pH range 4-7), and 6.0 g of Sequanal-grade urea. The solution was degassed and then 40 ~1 of Triton X- 100, 25 ~1 of 10% (w/v) ammonium persulfate, and 20 ~1 of TEMED were added, and the gel cast and the IO-well comb in place and allowed to polymerize for 1 h. After the gel had polymerized, the comb was removed and the wells were rinsed with distilled water. The wells and upper chamber
1
2
FIG. 2. A crude extract of Escherichia coli was analyzed by denaturing isoelectric focusing in the pH range 4-7 as described in the text and stained for protein with Coomassie blue. The pH gradient in the gel was measured and is indicated along the figure. Lane 1 contained 1.12 mg applied in 50 ~1, lane 2 contained 0.56 mg applied in 25 pl, and lane 3 contained 0.28 mg applied in 12 /.ll.
ET AL.
were filled with 25 IIIM H,P04 (anode solution) and the lower chamber was filled with 50 mM NaOH (cathode solution). The wells were then filled with 40 ~1 lysis buffer (8 M urea, 2% (v/v) ampholyte, 2% (v/v) Triton X-100, 1% (v/v) P-mercaptoethanol) diluted 1:4 with water. Samples were mixed with an equal volume of undiluted lysis buffer and then underlayed into the wells. The electrodes were connected so that the polarity was reversed, consistent with the electrode solutions. Electrophoresis was performed at room temperature, for 30 min at 150 V constant voltage, then increased to 200 V constant voltage for 2.5 h. After electrophoresis was complete, the gel was rinsed with distilled water and the pH gradient was measured as described above. Staining. After electrophoresis was complete, the gels were placed into 10% (v/v) trichloroacetic acid, gently shaken for 10 mitt, then transferred into 1% trichloroacetic acid for a minimum of 2 h to remove ampholytes. After a brief rinse with distilled water, gels were stained with 0.25% (w/v) Coomassie blue R-250 in 45% (v/v) methanol and 10% (v/v) glacial acetic acid for 10 min with gentle shaking. Excess stain was rinsed off with distilled water and the gel was destained in 45% (v/v) methanol and 10% (v/v) glacial acetic acid over activated charcoal. Two-dimensional electrophoresis. A crude sonic extract of Escherichia coli was analyzed by isoelectric focusing in the presence of 8 M urea, as previously described under Denaturing IEF, except that the gel was cast 1.O mm thick. The gel was fixed, stained, and the lane of interest was excised and equilibrated in SDS sample buffer for 15-30 min (1). Analysis in the second dimension was performed essentially as described by O’Farrell (1) except that the IEF gel slice was placed in direct contact with the stacking gel of the SDS system without the use of agarose. The SDS gel was cast in the vertical minigel system with an 8 X 7-cm cell format, 1.5 mm thick. Electrophoresis was performed at 150 V constant voltage until the dye front
293
FIG. 3. Two-dimensional analysis of a crude extract of Escherichia coli was performed as described in the text and stained for protein with Coomassie blue. Isoelectric focusing was performed in the pH range 4-7 in the presence of 8 M urea and the second dimension was SDS-polyacrylamide gel electrophoresis. The lane excised from the isoelectric focusing gel for subsequent analysis contained 2.24 mg of protein applied in 40 ~1.
reached the bottom of the gel, approximately 1 h. The two-dimensional gel was then stained with Coomassie blue R-250 as described above. RESULTS AND DISCUSSION
The gel shown in Fig. 1A demonstrates the resolution of protein standards in a gel containing pH 3 to 10 ampholytes. The pH gradient of the gel surface was determined by measurement with a flat bottom pH electrode and is indicated along the side of the figure. The pH gradient was linear in the pH range 4-9 (data not shown). The graph of reported p1 values versus migration distance,
displayed in Fig. lB, indicates that the pH gradient had been established in the gel and that the protein standards migrated to their isoelectric point. As can be seen in Fig. 2, the proteins in the crude sonic extract obtained from E. coli are well resolved in a gel containing 8 M urea, in the pH range 4 to 7. The surface pH profile was determined as previously described and is indicated along the side of the gel. As demonstrated in Fig. 3, the resolution of the proteins in a crude sonic extract of E. coli clearly indicates that the procedure is readily applicable to two-dimensional electrophoresis. The extract was analyzed by isoelectric focusing in the pH range 4 to 7 and
294
ROBERTSON
by SDS-polyacrylamide gel electrophoresis in the second dimension. Utilization of the vertical minigel format allowed this two-dimensional analysis to be performed in 1 day. Although there have been other reports of vertical slab gel isoelectric focusing, they did not employ the minigel system utilized here, and required either long run times or high voltage with special cooling (3,4). The procedure described here allows application of larger sample volumes (up to 50 ~1) containing higher concentrations of protein (up to 2 mg per lane) than commonly used in horizontal isoelectric focusing systems (2). The vertical minislab system has several advantages over tube gel systems. Multiple samples can be analyzed on the same slab gel, which is beneficial when comparing two-dimensional gels. Slab gels can be further analyzed by blotting techniques and easily dried for subsequent analysis by autoradiography. In addition, the time required for focusing in the vertical minislab is much less than most tube gel systems which require 12-40 h (1,5,6). Also, in contrast to other methods (1,4,5,7), prefocusing is not necessary with this system. Finally, this procedure has been successfully performed in several commer-
ET AL.
cially available minigel apparatuses, including those produced by Bio-Rad, Hoefer, and Idea Scientific. This report describes a method for the rapid resolution of protein mixtures utilizing an inexpensive system, which provides reproducible results in the presence or absence of urea, in both broad and narrow pH ranges and is readily applicable to two-dimensional electrophoresis. ACKNOWLEDGMENT This research was supported by Grant DMB-84 1454 from the National Science Foundation.
REFERENCES 1. O’Farrell, P. H. (1975) J. Biol. C/rem. 10, 4007-402 1. 2. Saravis, C. A., and Zamcheck, N. (1979) .I. Immunol. Methods 29,91-96. 3. Ames, G. F., and Nikaido, K. (1976) Biochemistry 15,616-623. 4. Giulian, G. G., Moss, R. L., and Greaser, M. (I 984) Anal. Biochem. 142,421-436. 5. Duncan, R., and Hershey, J. W. B. (1984) Anal. Biochem. 138, 144-155. 6. Manabe, T., Tachi, K., Kohjima, K., and Okuyama, T. (1979) J. Biochem. 85,649-659. 7. Kamboh, M. L. (1985) Electrophoresis 6, 185-186.
BIOCHEMISTRY
Rapid Isoelectric
167,290-294
(1987)
Focusing in a Vertical Polyacrylamide
Minigel System
EUGENE F. ROBERTSON,H.KATHLEENDANNELLY,PETERJ.MALLOY, ANDHENRYC.REEVES Department of Botany and Microbiology, Arizona State University, Tempe. Arizona
85287
Received April 24, 1987 A rapid method is described for the resolution of proteins employing isoelectric focusing in a vertical polyacrylamide minigel system. Isoelectric focusing can be performed in only 3 h, utilizing low voltage, under either native conditions or denaturing conditions in the presence of 8 M urea. The procedure permits the application of larger sample volumes containing more protein than other isoelectric focusing procedures, and provides the additional advantages of slab gels over tube gels for analytical purposes. The procedure is also well adapted for use in two-dimensional electrophoretic techniques, making it possible to complete a two-dimensional gel in 1 day. 0 1987 Academic Press, Inc. KEY WORDS: isoelectric focusing; electrophoresis; two-dimensional electrophoresis; protein; polyacrylamide gel; minigel.
Isoelectric focusing is in itself a powerful technique for the resolution of complex protein mixtures and when used under denaturing conditions as part of a two-dimensional analysis as many as 1,100 proteins can be resolved (1). This paper introduces a novel procedure for isoelectric focusing in vertical polyacrylamide minigels with several advantages over other methods that serve the same purpose. Isoelectric focusing in tube gels presents difficulties in the analysis of results due to the cylindrical configuration of the gel and problems often associated with comparing protein patterns in multiple cast gels. These problems can be avoided by employing horizontal slab gel isoelectric focusing; however, this procedure is limited by the inability to apply large sample volumes containing high concentrations of protein and the necessity for an efficient cooling plate on which to place the gel during the run (2). Isoelectric focusing performed in standard vertical, rather than horizontal, gels allows for the application of larger sample volumes but requires either long running times or high voltages (3,4). The method described in this paper eliminates these often encoun0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
tered problems and combines the advantages of the three commonly used methods for isoelectric focusing. MATERIALS
AND METHODS
Materials. Electrophoresis grade acrylamide and bis-acrylamide, N,N,N’, N’-tetramethylenediamine (TEMED),’ and ammonium persulfate were obtained from Bio-Rad Laboratories. Coomassie brilliant blue R-250 and isoelectric focusing standards in the range 3.55 to 9.3 were from Sigma Chemical Co. Ampholytes, in the pH range 3 to 10 and 4 to 7, were from Serva Fine Biochemicals. Sequanal-grade urea was purchased from Pierce Chemical Co. Native IEF. A vertical minigel system with an 8 X 7-cm cell format was utilized. The gels were cast 1.5 mm thick from the following mixture: 7 ml water, 2 ml acrylamide mixture (30% (w/v) acrylamide, 1% (w/v) bis-acrylamide), 2.4 ml 50% (v/v) glycerol, ’ Abbreviations used: IEF, isoelectric focusing: SDS, sodium dodecyl sulfate, TEMED, N,N,N’,N’-tetramethylenediamine. 290
MINIGEL
ISOELECTRIC
291
FOCUSING
I 2 Migration
4 Distance
(cm)
6
FIG. 1. Protein standards were analyzed by isoelectric focusing in the pH range 3-10 as described in the text. The three lanes in (A) each contain 16 rg of Sigma IEF 3.55-9.3 standard protein mix, applied in 20 ~1. The pH gradient was measured with a flat bottom pH electrode on the gel surface, and is indicated along the side of (A). The gel was stained for protein with Coomassie blue. The reported isoelectric points of the standard proteins were plotted against their migration distance from the bottom of the sample wells, and the best fit line is shown in (B). Published pI values for standard proteins are as follows: amyloglucosidase, 3.55; trypsin inhibitor, 4.55; @-Iactoglobulin A, 5.13; bovine carbonic anhydrase, 5.85; human carbonic anhydrase, 6.57; myoglobin, 6.76,7.16*; lactic dehydrogenase, 8.3,8.4,8.55*; trypsinogen, 9.3*. (*Indicates not visible in (A)).
and 0.6 ml ampholyte (pH range 3-10). These components were mixed, degassed, and then 50 ~1 of 10% (w/v) ammonium persulfate and 20 ~1 TEMED were added. Gels were cast with a lo-well comb in place and allowed to polymerize for 1 h. The cathode solution was 25 mM NaOH and the anode solution 20 mM acetic acid. These solutions were cooled to 4°C prior to electrophoresis. After polymerization was complete, the comb was removed and the wells were rinsed and then filled with the cathode solution. Protein standards were
mixed with an equal volume of 60% (v/v) glycerol and 4% (v/v) ampholyte of the same pH range used to prepare the gel. Electrophoresis was performed at room temperature for 1.5 h at 200 V constant voltage, then increased to 400 V constant voltage for an additional 1.5 h. After electrophoresis was complete the gel was rinsed briefly with distilled water and the pH gradient was measured on the gel surface with a flat bottom pH electrode. Denaturing ZEF. Isoelectric focusing gels containing 8 M urea were cast in a vertical 8
292
ROBERTSON
X 7-cm cell format as above. The mixture for a 1.5-mm gel contained 2.7 ml water, 2.0 ml acrylamide mixture (see above), 2.4 ml 50% glycerol, 0.6 ml Serva ampholyte (pH range 4-7), and 6.0 g of Sequanal-grade urea. The solution was degassed and then 40 ~1 of Triton X- 100, 25 ~1 of 10% (w/v) ammonium persulfate, and 20 ~1 of TEMED were added, and the gel cast and the IO-well comb in place and allowed to polymerize for 1 h. After the gel had polymerized, the comb was removed and the wells were rinsed with distilled water. The wells and upper chamber
1
2
FIG. 2. A crude extract of Escherichia coli was analyzed by denaturing isoelectric focusing in the pH range 4-7 as described in the text and stained for protein with Coomassie blue. The pH gradient in the gel was measured and is indicated along the figure. Lane 1 contained 1.12 mg applied in 50 ~1, lane 2 contained 0.56 mg applied in 25 pl, and lane 3 contained 0.28 mg applied in 12 /.ll.
ET AL.
were filled with 25 IIIM H,P04 (anode solution) and the lower chamber was filled with 50 mM NaOH (cathode solution). The wells were then filled with 40 ~1 lysis buffer (8 M urea, 2% (v/v) ampholyte, 2% (v/v) Triton X-100, 1% (v/v) P-mercaptoethanol) diluted 1:4 with water. Samples were mixed with an equal volume of undiluted lysis buffer and then underlayed into the wells. The electrodes were connected so that the polarity was reversed, consistent with the electrode solutions. Electrophoresis was performed at room temperature, for 30 min at 150 V constant voltage, then increased to 200 V constant voltage for 2.5 h. After electrophoresis was complete, the gel was rinsed with distilled water and the pH gradient was measured as described above. Staining. After electrophoresis was complete, the gels were placed into 10% (v/v) trichloroacetic acid, gently shaken for 10 mitt, then transferred into 1% trichloroacetic acid for a minimum of 2 h to remove ampholytes. After a brief rinse with distilled water, gels were stained with 0.25% (w/v) Coomassie blue R-250 in 45% (v/v) methanol and 10% (v/v) glacial acetic acid for 10 min with gentle shaking. Excess stain was rinsed off with distilled water and the gel was destained in 45% (v/v) methanol and 10% (v/v) glacial acetic acid over activated charcoal. Two-dimensional electrophoresis. A crude sonic extract of Escherichia coli was analyzed by isoelectric focusing in the presence of 8 M urea, as previously described under Denaturing IEF, except that the gel was cast 1.O mm thick. The gel was fixed, stained, and the lane of interest was excised and equilibrated in SDS sample buffer for 15-30 min (1). Analysis in the second dimension was performed essentially as described by O’Farrell (1) except that the IEF gel slice was placed in direct contact with the stacking gel of the SDS system without the use of agarose. The SDS gel was cast in the vertical minigel system with an 8 X 7-cm cell format, 1.5 mm thick. Electrophoresis was performed at 150 V constant voltage until the dye front
293
FIG. 3. Two-dimensional analysis of a crude extract of Escherichia coli was performed as described in the text and stained for protein with Coomassie blue. Isoelectric focusing was performed in the pH range 4-7 in the presence of 8 M urea and the second dimension was SDS-polyacrylamide gel electrophoresis. The lane excised from the isoelectric focusing gel for subsequent analysis contained 2.24 mg of protein applied in 40 ~1.
reached the bottom of the gel, approximately 1 h. The two-dimensional gel was then stained with Coomassie blue R-250 as described above. RESULTS AND DISCUSSION
The gel shown in Fig. 1A demonstrates the resolution of protein standards in a gel containing pH 3 to 10 ampholytes. The pH gradient of the gel surface was determined by measurement with a flat bottom pH electrode and is indicated along the side of the figure. The pH gradient was linear in the pH range 4-9 (data not shown). The graph of reported p1 values versus migration distance,
displayed in Fig. lB, indicates that the pH gradient had been established in the gel and that the protein standards migrated to their isoelectric point. As can be seen in Fig. 2, the proteins in the crude sonic extract obtained from E. coli are well resolved in a gel containing 8 M urea, in the pH range 4 to 7. The surface pH profile was determined as previously described and is indicated along the side of the gel. As demonstrated in Fig. 3, the resolution of the proteins in a crude sonic extract of E. coli clearly indicates that the procedure is readily applicable to two-dimensional electrophoresis. The extract was analyzed by isoelectric focusing in the pH range 4 to 7 and
294
ROBERTSON
by SDS-polyacrylamide gel electrophoresis in the second dimension. Utilization of the vertical minigel format allowed this two-dimensional analysis to be performed in 1 day. Although there have been other reports of vertical slab gel isoelectric focusing, they did not employ the minigel system utilized here, and required either long run times or high voltage with special cooling (3,4). The procedure described here allows application of larger sample volumes (up to 50 ~1) containing higher concentrations of protein (up to 2 mg per lane) than commonly used in horizontal isoelectric focusing systems (2). The vertical minislab system has several advantages over tube gel systems. Multiple samples can be analyzed on the same slab gel, which is beneficial when comparing two-dimensional gels. Slab gels can be further analyzed by blotting techniques and easily dried for subsequent analysis by autoradiography. In addition, the time required for focusing in the vertical minislab is much less than most tube gel systems which require 12-40 h (1,5,6). Also, in contrast to other methods (1,4,5,7), prefocusing is not necessary with this system. Finally, this procedure has been successfully performed in several commer-
ET AL.
cially available minigel apparatuses, including those produced by Bio-Rad, Hoefer, and Idea Scientific. This report describes a method for the rapid resolution of protein mixtures utilizing an inexpensive system, which provides reproducible results in the presence or absence of urea, in both broad and narrow pH ranges and is readily applicable to two-dimensional electrophoresis. ACKNOWLEDGMENT This research was supported by Grant DMB-84 1454 from the National Science Foundation.
REFERENCES 1. O’Farrell, P. H. (1975) J. Biol. C/rem. 10, 4007-402 1. 2. Saravis, C. A., and Zamcheck, N. (1979) .I. Immunol. Methods 29,91-96. 3. Ames, G. F., and Nikaido, K. (1976) Biochemistry 15,616-623. 4. Giulian, G. G., Moss, R. L., and Greaser, M. (I 984) Anal. Biochem. 142,421-436. 5. Duncan, R., and Hershey, J. W. B. (1984) Anal. Biochem. 138, 144-155. 6. Manabe, T., Tachi, K., Kohjima, K., and Okuyama, T. (1979) J. Biochem. 85,649-659. 7. Kamboh, M. L. (1985) Electrophoresis 6, 185-186.