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A new discontinuous buffer system for the electrophoresis of cationic proteins at near-neutral pH
ANALYTICAL
BIOCHEMISTRY
118,
194-196 (1981)
A New Discontinuous of Cationic
Buffer System for the Electrophoresis Proteins at Near-Neutral pH
JOHN M. THOMAS AND M. E. HODES’ ~~~artme~t ~~~edie~I
Genetics, Indiana University School of Medicine, I~di~a~I~~,
Iliad
44223
Received August 10, 1981 A simple, discontinuous buffer system for polyacrylamide gel electrophoresis near neutral pH is described. The buffer is MOPS (3-(N-morpholine]propanesuIfonic acid), the leading ion K” and the trailing ion histidine. The system offers improved resolution of cationic proteins.
Discontinuous buffer systems for the efectrophoresis of cationic proteins at near-neutral pH have been described previously ( l3). One of these systems (3), however, does not employ stacking, which enables automatic sample concentration of dilute protein solutions (4), and the other two systems ( 1,2) utilize undesirable pyridine derivatives in the electrode buffer. We have sought a simple, d~continuous buffer system for polyacrylamide gel electrophoresis which would offer improved resolution of cationic proteins in comparison to that obtained with the commonly used acid disc electrophoresis system of Reisfeld et al. (5). This report describes a new electrophoresis system which stacks and separates near neutral pH and provides improved resolution of some common cationic proteins. MATERIALS AND METHODS
Myoglobin (from sperm whale skeletal muscle), cytochrome c (from horse heart), r.-histidine and @alanine were obtained from Sigma Chemical Company. MOPS* 3-(Nmorpholino)propanesulfonic acid, was purchased from Calbi~hem. Electrophoresis was performed using an apparatus modified I To whom all correspondence should be addressed. * Abbreviation used: MOPS, 3-(N-morpho1ino)propanesulfonic acid. 0003-2697/8 I/ 170194-03$02.~/0 Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any fom resewed.
from the design of Studier (6). The thinlayer polyacrylamide gels were 1.5 mm thick, and the separating gels were 11 cm long. The acid disc electrophoresis system (system A) used for comparison was modified from that of Reisfeld et al. (51, and employs K+ as the leading ion, @-alanine as the trailing ion, and acetic acid as the buffer. In the newly designed neutral disc electrophoresis system (system B), K+ is the leading ion, histidine is the trailing ion, and MOPS is the buffer. The composition of the two electrophoresis systems is compared in Table 1. All gels were photopolymerized to avoid deleterious effects from persulfate. RESULTS AND DISCUSSION
Two colored proteins, myoglobin and cytochrome c, frequently used as marker proteins in cathodally migrating systems, were used to compare resolution obtained with the two systems. These samples were diluted with buffered glycerol and applied to sample wells as described elsewhere (8). The electrophoretic runs were in a constant current mode, starting at 65 V (about 20 mA) and lasting 3.5 h for gel system A, and starting at 80 V (about 12 mA) and lasting 5 h for gel system B. The duration of the runs was based on the migration distance of the major band of cytochrome c. The protein bands 194
NEW
ELECTROPHORESIS
195
BUFFER
TABLE
1
COMPOSITION OF GELS AND ELECTRODE BUFFERS System Bb
System A” Stacking gel PH Acrylamide N,N-Methylenebisacrylamide Glycerol
6.5 3.2%’ (T = 4.0%)d 0.8% (C = 20.0%)d 20.0%
KOH TEMED’
0.06 0.05%
Acetic acid/
0.06 M
M
0.10%
0.063 M
MOPS’
Riboflavin Riboflavin S-phosphate Separating gel PH Acrylamide NJ’-Methylenebisacrylamide KOH TEMED
Acetic acid’ MOPS’ Riboflavin Electrode buffer PH @-Alanine Acetic acid Histidine
8.0 3.2% (7’ = 4.0%) 0.8% (C = 20.0%) 20.0% 0.05 M
0.005% 0.0005% 4.5 9.7% (T = 10.0%) 0.3% (C = 3.0%)
6.8 9.7% (T = 10.0%) 0.3% (C = 3.0%)
0.06 0.05%
0.05 0.05%
M
M
0.16 M 0.0005% 4.5 3.12% (0.35 M) 0.80% (0.14 M)
MOPS
0.213 M 0.0005% 6.8 1.56% (0.10 M) 0.46% (0.022 M)
@Modified from Reisfeld ez 01. (5). ’ Newly designed disc electrophoresis system operating near neutral pH. ’ Percentage values are (w/v), except glycerol, acetic acid, and TEMED (v/v). d Definition of T and C (7). ’ N.N,N,N”-tetramethylethylenediamine. ‘Acetic acid or MOPS is added to KOH/T~MED in a 4X con~ntrated stock buffer to give the appropriate PH.
were stained with Coomassie brilliant blue R-250 as described elsewhere (8). The improved resolution, obtained with the newly designed neutral disc electrophoresis system, is illustrated in Fig. 1 for two common cationic proteins, myoglobin and cytochrome c, having isoelectric points of 7.7 to 8.2 and 9.0 to 9.4, respectively (9). Multiple protein bands barely separated at acid pH are clearly separated at a pH nearer neutrality and nearer the isoelectric points of the proteins-in accordance with the theory of electrophoresis of proteins ( 10). This new discontinuous buffer system for
the electrophoresis of cationic proteins near neutral pH was designed by using the general guidelines for disc electrophoresis suggested by Williams and Reisfeld ( 11). The following specifications ( 11) are satisfied in this new system: (a) the trailing ion (histidine, p& = 6.0) is a weak base with p& up to 1 pH unit less than the pH of the separating gel (pH 6.8) (b) the pK, of the buffer (MOPS, pK, 7.2) is up to 1 pH unit higher than that of the separating gel (pH 6.8) and (c) the pH of the stacking gel (pH 8.0) is between 2 and 3 pH units more than the pK, of the trailing ion (histidine, p& 6.0). This
THOMAS
B 3
12
3
AND
HODES
uous electrophoresis systems by following the general criteria rather than resorting to the computer-generated systems (12) derived from more complicated theory. ACKNOWLEDGMENT This is publication No. 81-02 of the Department of Medical Genetics.
REFERENCES
0 FIG. 1. Comparison of the resolution obtained with system A, the commonly used acid disc electrophoresis system of Reisfeld et al. (5), and system B, a newly designed disc electrophoresis system operating near neutral pH. Samples (200 pg each): 1, myoglobin; 2, cytochrome c; and 3, mixture of myoglobin and cytochrome c.
5. 6. 7. 8. 9. 10.
system employing MOPS as the buffer is not included in an extensive, computer-derived compilation of buffer recipes for “multiphasic zone electrophoresis” ( 12). Thus, it is possible to design useful, new discontin-
11. 12.
Taber, H. W., and Sherman, F. (1964) Ann N. Y. Acad. Sci. 121,600-615. Young, C. W., Dessources, W., Hodas, S., and Bittar, E. S. (1975) Cancer Res. 35, 1991-1995. Hoffmann, P. J., and Chalkley, R. (1976) Anal. Biochem. 76, 539-546. Chen, B., Griffith, A., Catsimpoolas, N., Chrambath, A., and Rodbard, D. (1978) Anal. Biochem. 89, 609-6 15. Reisfeld, R. A., Lewis, U. J., and Williams, D. E. (1962) Nature (London) 195, 281-283. Studier, F. W. (1973) J. Mol. Biol. 79, 237-248. Hjertin, S. ( 1962) Arch. Biochem. 5iophy.s. Suppl 1, 147-151. Thomas, J. M., and Hodes, M. E. ( 1.981) Electrophoresis 2, 104- 112. Righetti, P. G., and Caravaggio, T. (1976) J. Chromatogr. 127, l-28. Mauer, H. R. (1971) Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, 2nd ed., de Gruyter, Berlin. Williams, D. E., and Reisfeld, R. A. (1964) Ann. N. Y. Acad. Sci. 121, 373-381. Jovin, T. M. (1973) Ann. N. K Acad. Sci. 209, 477-496.
BIOCHEMISTRY
118,
194-196 (1981)
A New Discontinuous of Cationic
Buffer System for the Electrophoresis Proteins at Near-Neutral pH
JOHN M. THOMAS AND M. E. HODES’ ~~~artme~t ~~~edie~I
Genetics, Indiana University School of Medicine, I~di~a~I~~,
Iliad
44223
Received August 10, 1981 A simple, discontinuous buffer system for polyacrylamide gel electrophoresis near neutral pH is described. The buffer is MOPS (3-(N-morpholine]propanesuIfonic acid), the leading ion K” and the trailing ion histidine. The system offers improved resolution of cationic proteins.
Discontinuous buffer systems for the efectrophoresis of cationic proteins at near-neutral pH have been described previously ( l3). One of these systems (3), however, does not employ stacking, which enables automatic sample concentration of dilute protein solutions (4), and the other two systems ( 1,2) utilize undesirable pyridine derivatives in the electrode buffer. We have sought a simple, d~continuous buffer system for polyacrylamide gel electrophoresis which would offer improved resolution of cationic proteins in comparison to that obtained with the commonly used acid disc electrophoresis system of Reisfeld et al. (5). This report describes a new electrophoresis system which stacks and separates near neutral pH and provides improved resolution of some common cationic proteins. MATERIALS AND METHODS
Myoglobin (from sperm whale skeletal muscle), cytochrome c (from horse heart), r.-histidine and @alanine were obtained from Sigma Chemical Company. MOPS* 3-(Nmorpholino)propanesulfonic acid, was purchased from Calbi~hem. Electrophoresis was performed using an apparatus modified I To whom all correspondence should be addressed. * Abbreviation used: MOPS, 3-(N-morpho1ino)propanesulfonic acid. 0003-2697/8 I/ 170194-03$02.~/0 Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any fom resewed.
from the design of Studier (6). The thinlayer polyacrylamide gels were 1.5 mm thick, and the separating gels were 11 cm long. The acid disc electrophoresis system (system A) used for comparison was modified from that of Reisfeld et al. (51, and employs K+ as the leading ion, @-alanine as the trailing ion, and acetic acid as the buffer. In the newly designed neutral disc electrophoresis system (system B), K+ is the leading ion, histidine is the trailing ion, and MOPS is the buffer. The composition of the two electrophoresis systems is compared in Table 1. All gels were photopolymerized to avoid deleterious effects from persulfate. RESULTS AND DISCUSSION
Two colored proteins, myoglobin and cytochrome c, frequently used as marker proteins in cathodally migrating systems, were used to compare resolution obtained with the two systems. These samples were diluted with buffered glycerol and applied to sample wells as described elsewhere (8). The electrophoretic runs were in a constant current mode, starting at 65 V (about 20 mA) and lasting 3.5 h for gel system A, and starting at 80 V (about 12 mA) and lasting 5 h for gel system B. The duration of the runs was based on the migration distance of the major band of cytochrome c. The protein bands 194
NEW
ELECTROPHORESIS
195
BUFFER
TABLE
1
COMPOSITION OF GELS AND ELECTRODE BUFFERS System Bb
System A” Stacking gel PH Acrylamide N,N-Methylenebisacrylamide Glycerol
6.5 3.2%’ (T = 4.0%)d 0.8% (C = 20.0%)d 20.0%
KOH TEMED’
0.06 0.05%
Acetic acid/
0.06 M
M
0.10%
0.063 M
MOPS’
Riboflavin Riboflavin S-phosphate Separating gel PH Acrylamide NJ’-Methylenebisacrylamide KOH TEMED
Acetic acid’ MOPS’ Riboflavin Electrode buffer PH @-Alanine Acetic acid Histidine
8.0 3.2% (7’ = 4.0%) 0.8% (C = 20.0%) 20.0% 0.05 M
0.005% 0.0005% 4.5 9.7% (T = 10.0%) 0.3% (C = 3.0%)
6.8 9.7% (T = 10.0%) 0.3% (C = 3.0%)
0.06 0.05%
0.05 0.05%
M
M
0.16 M 0.0005% 4.5 3.12% (0.35 M) 0.80% (0.14 M)
MOPS
0.213 M 0.0005% 6.8 1.56% (0.10 M) 0.46% (0.022 M)
@Modified from Reisfeld ez 01. (5). ’ Newly designed disc electrophoresis system operating near neutral pH. ’ Percentage values are (w/v), except glycerol, acetic acid, and TEMED (v/v). d Definition of T and C (7). ’ N.N,N,N”-tetramethylethylenediamine. ‘Acetic acid or MOPS is added to KOH/T~MED in a 4X con~ntrated stock buffer to give the appropriate PH.
were stained with Coomassie brilliant blue R-250 as described elsewhere (8). The improved resolution, obtained with the newly designed neutral disc electrophoresis system, is illustrated in Fig. 1 for two common cationic proteins, myoglobin and cytochrome c, having isoelectric points of 7.7 to 8.2 and 9.0 to 9.4, respectively (9). Multiple protein bands barely separated at acid pH are clearly separated at a pH nearer neutrality and nearer the isoelectric points of the proteins-in accordance with the theory of electrophoresis of proteins ( 10). This new discontinuous buffer system for
the electrophoresis of cationic proteins near neutral pH was designed by using the general guidelines for disc electrophoresis suggested by Williams and Reisfeld ( 11). The following specifications ( 11) are satisfied in this new system: (a) the trailing ion (histidine, p& = 6.0) is a weak base with p& up to 1 pH unit less than the pH of the separating gel (pH 6.8) (b) the pK, of the buffer (MOPS, pK, 7.2) is up to 1 pH unit higher than that of the separating gel (pH 6.8) and (c) the pH of the stacking gel (pH 8.0) is between 2 and 3 pH units more than the pK, of the trailing ion (histidine, p& 6.0). This
THOMAS
B 3
12
3
AND
HODES
uous electrophoresis systems by following the general criteria rather than resorting to the computer-generated systems (12) derived from more complicated theory. ACKNOWLEDGMENT This is publication No. 81-02 of the Department of Medical Genetics.
REFERENCES
0 FIG. 1. Comparison of the resolution obtained with system A, the commonly used acid disc electrophoresis system of Reisfeld et al. (5), and system B, a newly designed disc electrophoresis system operating near neutral pH. Samples (200 pg each): 1, myoglobin; 2, cytochrome c; and 3, mixture of myoglobin and cytochrome c.
5. 6. 7. 8. 9. 10.
system employing MOPS as the buffer is not included in an extensive, computer-derived compilation of buffer recipes for “multiphasic zone electrophoresis” ( 12). Thus, it is possible to design useful, new discontin-
11. 12.
Taber, H. W., and Sherman, F. (1964) Ann N. Y. Acad. Sci. 121,600-615. Young, C. W., Dessources, W., Hodas, S., and Bittar, E. S. (1975) Cancer Res. 35, 1991-1995. Hoffmann, P. J., and Chalkley, R. (1976) Anal. Biochem. 76, 539-546. Chen, B., Griffith, A., Catsimpoolas, N., Chrambath, A., and Rodbard, D. (1978) Anal. Biochem. 89, 609-6 15. Reisfeld, R. A., Lewis, U. J., and Williams, D. E. (1962) Nature (London) 195, 281-283. Studier, F. W. (1973) J. Mol. Biol. 79, 237-248. Hjertin, S. ( 1962) Arch. Biochem. 5iophy.s. Suppl 1, 147-151. Thomas, J. M., and Hodes, M. E. ( 1.981) Electrophoresis 2, 104- 112. Righetti, P. G., and Caravaggio, T. (1976) J. Chromatogr. 127, l-28. Mauer, H. R. (1971) Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, 2nd ed., de Gruyter, Berlin. Williams, D. E., and Reisfeld, R. A. (1964) Ann. N. Y. Acad. Sci. 121, 373-381. Jovin, T. M. (1973) Ann. N. K Acad. Sci. 209, 477-496.