- Email: [email protected]
A simplified gel electrophoretic system and its validity for molecular weight determinations of protein-cetyltrimethylammonium complexes
ANALYTICAL
BIOCHEMISTRY
81, 320-327 (1977)
A Simplified Gel Electrophoretic System and Its Validity for Molecular Weight Determinations of ProteinCetyltrimethylammonium Complexes SAKOL PANYIM,RANGSAN AND DAUNGRUDEE Department
THITIPONGPANICH, SUPATIMUSRO
of Biochemistry, Faculty of Science, Mahidol Rama VI Road. Bangkok, Thailand
University,
Received November 3, 1976: accepted April 14, 1977 Proteins ranging in molecular weight from 90.000 to 15,000 daltons were studied by polyacrylamide gel electrophoresis in the presence of cetyltrimethylammonium bromide (CTAB) at pH 4.6. The results showed a linear relationship between log molecular weight and relative electrophoretic mobility. The simplicity and reliability of the method offer an alternative means of determination of molecular weights of a wide variety of proteins by gel electrophoresis.
SDS-Gel electrophoresis has been widely used for determining molecular weights of a wide variety of proteins since its introduction by Shapiro et al. (1) and subsequent careful analysis by Weber and Osborn (2). However, highly basic proteins are sparingly soluble in SDS, and this makes their analysis on SDS-electrophoresis unreliable. Moreover, nucleic acids are soluble in SDS, and, often, their presence complicates SDS-electrophoretic analysis of proteins. We recently employed SDSpolyacrylamide gel electrophoresis to study phosphorylation products of protein kinases in spermatozoa and encountered interfernce by nucleic acids. We employed cetyltrimethylammonium bromide (CTAB) to precipitate the nucleic acid and to perform electrophoretic analyses of the proteins successfully. The recent report by Schick (3) that polyacrylamide gel electrophoreses of nine selected proteins in the presence of N-cetylpyridinium chloride gave a linear relationship between log molecular weight and the relative mobility prompted us to test if electrophoresis in the presence of CTAB would also give such a relationship. Our careful analyses did show a linear relationship between log molecular weights and electrophoretic mobilities of a wide variety of proteins. Moreover, we found our procedure less time consuming and simpler than that in the previous report (3). 320 CopyrIght All rights
0 1977 by Academx Pres,. Inc. of reproductmn I” any form reserved.
ISSN 0003-2697
CTAB-GEL
MATERIALS
ELECTROPHORESIS
321
AND METHODS
Chemicals and standard proteins. Analytical-grade sodium acetate and glacial acetic acid were obtained from J. T. Baker Chemical Co. Acrylamide (electrophoresis grade), methylenebisacrylamide, and N,N,N’.N’-tetramethylethylenediamine were from Eastman. Cetyltrimethylammonium bromide (CTAB), Coomassie brilliant blue R-250, and ammonium persulfate were obtained from Sigma. Urea was from Schwarz-Mann, and 2-mercaptoethanol was from Calbiochem. The standard proteins obtained from Sigma were: adolase (rabbit muscle), pyruvate kinase (rabbit skeleton), fructose-6-phosphate kinase (rabbit muscle), y-globulin, ovalbumin, myoglobin, hemoglobin, and lactic dehydrogenase (rabbit muscle). cr-Amylase and carboxypeptidase A were from Worthington. Bovine serum albumin was from Calbiochem. D-Amino acid oxidase (pig kidney) was from Schwarz Bioresearch. Phosphorylase B was from Boeringer Mannheim. Concanavalin A was from Miles Laboratories. Preparation ofprotein Samples. All standard proteins were dissolved at approximately 0.25-0.5 mgiml in 0.01 M sodium acetate, pH 4.6, 0.5% CTAB, and 1.0% 2-mercaptoethanol and were boiled for 5 min or incubated at 37°C for 2 hr. At the end of the boiling or incubation period. solid urea was added to give a final concentration of 6.0 M. After approximately 30 min of further incubation, the protein solutions were ready for loading onto the gel. Preparation of polyacrylamide gels and electrode buffer. Solution A: 30.0 g% acrylamide, 0.45 g% methylene bisacrylamide. Solution B: 245 ml of 0.2 M acetic acid, 355 ml of 0.2 M sodium acetate, 2 g of CTAB, and water to a volume of 1000 ml. Solution C: 2.5 mgiml of ammonium persulfate. For making 10% acrylamide gel in a total of 12 gel tubes (6.0 mm i.d. and 10 cm long), 12 ml of solution A, 16.2 ml of solution B, 1.8 ml of solution C, 6 ml of water, and 0.12 ml of TEMED were thoroughly mixed. The solution was poured into the glass tubes, and water was carefully overlayered to flatten the gel top. All solutions and the above operations were at room temperature. The filled gel tubes were then placed in a 37°C oven, and the polymerization took about 15 min. For 7.5 and 5% acrylamide gels, the preparation was as described above, except the amounts of solution A and water were varied accordingly . The buffer in the electrode compartments was prepared by adding an equal volume of water to solution B. Normally, 2-3 liters were required for electrophoresis in the ISCO Model 419 electrophoresis apparatus. Sample loading and electrophoresis. Twenty microliters of protein solution, prepared as above, was normally loaded onto gels. Azure A dissolved in 0.01 M sodium acetate, pH 4.6, and 10% sucrose (0.5 mgiml)
322
FIG.
1.47%
PANYIM.
THITIPONGPANICH.
1. Electrophoretic pattern cross-linker bisacrylamide.
AND
SUPATIMUSRO
of protein-CTAB complexes in 10% acrylamide Numbers represent the protein code in Table
gel with 1.
was used as a marker dye. Normally, 10 ~1 of the marker dye was electrophoresed for about 3 min prior to sample loading. This was to prevent contact between the Azure A and the protein solution and to sharpen the marker dye boundary. Electrophoresis was performed at constant values of 80- 100 V and S- 10 mA/gel until the Azure A moved close to the bottom of gel tube. The Azure A position was marked by inserting a small wire at the middle of the band. The current tended to increase about 10% at the end of the run. Staining and destaining. The staining solution was that described by Weber and Osborn (2) containing 1.25 g of Coomassie brilliant blue dissolved in 454 ml of 50% methanol and 46 ml of glacial acetic acid. Staining was performed at 80-100°C for 30-60 min. Destaining was done in large volumes of 0.9 N acetic acid at 80-100°C. With frequent changes of 0.9 N acetic acid, all stained bands could be visible in 2-3 hr. Excessively long exposure to hot acetic acid may lead to slow bleaching of stained bands. The gels were stored at room temperature in 0.9 N acetic acid. The mobilities of proteins and Azure A were measured after destain-
CTAB-GEL
TABLE MOLECULAR
WEIGHTS
323
ELECTROPHORESIS
OF PROTEIN
I SUB~NIB
.AND
THEIR
CODES
Subunit molecular weight Code I 2 3 3
Protein
(daltons)
Phosphorylase B Fructose h-phosphate Bovine serum albumin
kinase
Reference
92,500 7x.000 68 .ooo
12 II 7
5 6
Pyruvate kinase Heavy chain y-globulin Ovalbumin
j7.000 50.000 43 .ooo
I2 2 ?
7 x
PIdolase D-Amino
40.000 3 7 .ooo
2 7
?S.OOO 35 .ooo 34.600
2 I? 2
17,000 25,000
I2 I’
17200 15.500
7 2
acid
oxidase
9 IO II
Pepsin Lactic dehydrogenase Carboxypeptidase
I! I3
Concanavalin
13 IS
A
A
u-Amylase Myoglobin Hemoglobin
ing, and relative mobility was obtained protein by that of the dye. RESULTS
by dividing
the mobility
of the
AND DISCUSSION
By converting globular proteins to rod-like structures with approximately equal charge per unit weight, SDS made these proteins separable in SDS-polyacrylamide gel electrophoresis on the basis of size (2,4). CTAB. a cationic detergent, should similarly change a globular protein to one with a rod-like structure and, therefore. should be equally useful in estimating protein molecular weights. Unfortunately, there were many technical problems associated with CTAB-polyacrylamide gel electrophoresis, namely, uniform polymerization of the gel and staining, and destaining procedures. CTAB tended to form precipitates during gel polymerization and, thus, made the gel structure variable. CTAB bound and precipitated the Coomassie blue and had to be removed prior to successful staining. To surmount these problems, we polymerized the acrylamide at 37°C to obtain a uniform gel structure and performed staining and desgianing at 80- 100°C to prevent precipitation of the CTAB complex and to accelerate the tasks. The results, as shown in Fig. 1. demonstrate good resolution of proteins ranging in molecular weight from 17,000 to 68,000 daltons. It is evident that the electrophoretic mobilities are in the order: myoglobin > concanavalin A > lactic dehydrogenase > adolase > ovalbumin > pyruvate kinase > bovine serum albumin. After examining the molecular weights of these protein subunits (Table 1). it
324
PANYIM.
THITIPONGPANICH,
AND
SUPATIMUSRO
lo-
0.3
0.4
0.5
Relative
0.6
0.7
mobility
FIG. 2. A linear relationship between log molecular weights and relative electrophoretic mobilities. The electrophoresis was performed in 10% acrylamide gel with 1.47% crosslinker. Numbers denote the protein code in Table 1.
becomes apparent that their electrophoretic mobilities vary directly with the subunit molecular weights. This is more clearly seen in Fig. 2, in which relative electrophoretic mobilities are plotted against their log molecular weights; a linear relationship between these two parameters was observed. Figure 2 shows the linear relationship within the molecular weight range of 15,000-70,000 daltons, using 10% acrylamide gel with 1.47% crosslinker at pH 4.6. Increasing the amount of cross-linker alone, as seen in
0.2
0.4 Relative
II.6
mobility
FIG. 3. Dependence of electrophoretic mobility on the amount of cross-linker. trophoresis was performed in a constant 10% acrylamide gel with 1.47% (right curve), (middle curve), and 5.67% (left curve) cross-linker.
Elec2.90%
CTAB-GEL
325
ELECTROPHORESIS
FIG. 4. Dependence of electrophoretic mobility on acrylamide phoresis was performed in a constant 1.47% cross-linker with (middle curve), and 10% (left curve) acrylamide.
concentration. Eiectro5% (right curve). 7.5%
the Fig. 3, leads to deviation from the linearity. At 5.6% cross-linker, the linearity extends from 15,000 to about 40,000 daltons. Changing acrylamide concentration at a constantly low cross-linker concentration, as shown in the Fig. 4, does not lead to deviation from linearity. A linear relationship of log molecular weights and relative mobilities is observed in all three acrylamide concentrations. Acrylamide concentration is known to alter lengths of gel fibers per unit volume, while the cross-linker, methylenebisacrylamide, changed the thickness of the gel fibers (5). Apparently, the linear relationship between log molecular I
0
2.5
5.0
7.5 % acrylamide
10.0
FIG. 5. Relationship between log relative mobility and acrylamide concentration constant 1.47% cross-linker. Numbers represent the protein code in Table 1.
in a
326
PANYIM.
THITIPONGPANICH.
4
:’
AND
SUPATIMUSRO
,/:.;
2
L
6
a
10
KrxlOO
FIG. 6. Plot of retardation coefficient (K,) versus molecular weight for seven proteins. The retardation coefficient was computed from the data in Fig. 5.
standard
weight and relative mobility in the CTAB-polyacrylamide gel system is more sensitive to alteration in the thickness than in the length of gel fibers. The results indicate that the structure of the gel pore is a determining factor in the linear relationship of log molecular weight and relative mobility of proteins. Earlier investigators found that CTAB-polyacrylamide gel gives a linear relationship over a short molecular weight range of lO,OOO-20,000 daltons (6). However, optimal conditions (e.g.. cross-linker, acrylamide concentration, or pH) were not explored. When log relative mobility is plotted against acrylamide concentration at a constant cross-linker concentration, as shown in Fig. 5, a series of linear relationships, as predicted by Ferguson’s ‘equation (7), are obtained. Upon extrapolation, all lines intersect approximately at zero acrylamide concentration, indicating that these proteins move at comparable mobilities in free solution (5,8). Therefore, the CTAB-bound proteins should be fractionated in CTAB-polyacrylamide gel electrophoresis solely on basis of size difference (8,9). In SDS-polyacrylamide gel electrophoresis, in which separation was shown to be based on size, SDS-bound proteins were similarly shown to have comparable mobilities in the free solution (9- 11). When molecular weight is plotted against its retardation coefficient, as shown in Fig. 6, a parabolic curve could be best drawn through these points. Frank and Rodbard (11) had demonstrated that the plot of molecular weight versus its retardation coefficient in SDSgel electrophoresis yielded a parabola. Schick (3) had previously shown that N-cetylpyridinium chloride could convert the electrophoretic mobilities of six selected proteins to become linearly related to their molecular weights. Such an electrophoretic system, using 10% acrylamide, 2.70% cross-linker. was claimed to yield a
CTAB-GEL
37-7
ELECTROPHORESIS
linear relationship in a molecular weight range from 17,000 to 170,000 daltons, even without reduction of disulfide linkages. Our present system, under a comparable condition ( 10% acrylamide, 2.90% cross-linker), gives the linear relationship only from 15,000 to 40,000 daltons, or to 70,000 daltons if the cross-linker is reduced to 1.47%. Although our system and Schick’s system differ in the structure of cationic detergents, in pH, and in initiator for gel polymerization, it is difficult to explain the different extent of linearity on these bases. Neville (9) had earlier pointed out the sigmoidal relationship of log molecular weight and relative mobility of the protein-SDS complex and had further demonstrated that a linear relationship could be attenable in the molecular weight range of only 15,00070.000 daltons using comparable acrylamide and cross-linker concentrations. It is noteworthy that our procedure takes 3 hr for electrophoresis, 1 hr for staining, and 2-3 hrs for destaining: therefore, the analysis is not time-consuming and can be accomplished in less than 10 hr. In addition, the procedure is greatly simplified and, consequently, less tedious. As pointed out earlier, CTAB precipitates nucleic acids (13), whereas SDS does not, therefore, the CTAB electrophoresis should allow analysis of proteins in the presence of nucleic acids. For example, an analysis of the total chromosomal proteins of nucleus, without prior removal of DNA, could be envisaged. ACKNOWLEDGMENTS We ceived Health
wish to thank Dr. Bhinyo Panijpan for his valuable financial support from the Faculty of Science, Mahidol Organization.
discussion. University,
This work reand the World
REFERENCES I. Shapiro, A. L., Vinuela. E. and Maizel, J. V.. Jr. (1967) Biochem. Biophy~. Rrs. Commun. 28, 815-820. 2. Weber, K., and Osborn, M. (1970) J. Bid. Chem. 244. 4406-4412. 3. Schick, M. (1975) Anal. Biochem. 63, 345-349. 4. Reynolds, J. A., and Tanford. C. (197O)J. Bid. C17rm. 245, 5161-5165. 5. Chrambach, A., and Rodbard. D. (1971) Science 172, 440-451. 6. Williams, J. G.. and Gratzer. W. B. (1971) J. Chromntogr. 57, 121-125. 7. Ferguson, K. A. (1964) Metabolism 13, 985- 1002. 8. Hedrick, J., and Smith, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. 9. Neville, D. M., Jr. (1971) J. Bid. Chrm. 246, 6338-6334. 10. Weber. K., and Osborn. M. (1975) in The Proteins. Neurath. H.. and Hill. R. L.. eds.). 3rd ed.. Vol. 1, pp. 179-223. Academic Press, New York. Il. Frank, R. N.. and Rodbard. D. (1975) Arch. Biochrm. Biophys. 171, I-13. 12. Klotz, I. M., Darnall, E. W.. and Langerma. N. R. (1975) in The Proteins. (Neurath. H.. and Hill, R. L.. eds.). 3rd ed.. Vol. 1, pp. 293-411, Academic Press, New York. 13. Reitz. M. S., Jr.. Abrell. J. W.. Trainor, C. D.. and Gallo, R. C. (1972) Biothem. Biophys. Res. Commun. 49, 30-38.
BIOCHEMISTRY
81, 320-327 (1977)
A Simplified Gel Electrophoretic System and Its Validity for Molecular Weight Determinations of ProteinCetyltrimethylammonium Complexes SAKOL PANYIM,RANGSAN AND DAUNGRUDEE Department
THITIPONGPANICH, SUPATIMUSRO
of Biochemistry, Faculty of Science, Mahidol Rama VI Road. Bangkok, Thailand
University,
Received November 3, 1976: accepted April 14, 1977 Proteins ranging in molecular weight from 90.000 to 15,000 daltons were studied by polyacrylamide gel electrophoresis in the presence of cetyltrimethylammonium bromide (CTAB) at pH 4.6. The results showed a linear relationship between log molecular weight and relative electrophoretic mobility. The simplicity and reliability of the method offer an alternative means of determination of molecular weights of a wide variety of proteins by gel electrophoresis.
SDS-Gel electrophoresis has been widely used for determining molecular weights of a wide variety of proteins since its introduction by Shapiro et al. (1) and subsequent careful analysis by Weber and Osborn (2). However, highly basic proteins are sparingly soluble in SDS, and this makes their analysis on SDS-electrophoresis unreliable. Moreover, nucleic acids are soluble in SDS, and, often, their presence complicates SDS-electrophoretic analysis of proteins. We recently employed SDSpolyacrylamide gel electrophoresis to study phosphorylation products of protein kinases in spermatozoa and encountered interfernce by nucleic acids. We employed cetyltrimethylammonium bromide (CTAB) to precipitate the nucleic acid and to perform electrophoretic analyses of the proteins successfully. The recent report by Schick (3) that polyacrylamide gel electrophoreses of nine selected proteins in the presence of N-cetylpyridinium chloride gave a linear relationship between log molecular weight and the relative mobility prompted us to test if electrophoresis in the presence of CTAB would also give such a relationship. Our careful analyses did show a linear relationship between log molecular weights and electrophoretic mobilities of a wide variety of proteins. Moreover, we found our procedure less time consuming and simpler than that in the previous report (3). 320 CopyrIght All rights
0 1977 by Academx Pres,. Inc. of reproductmn I” any form reserved.
ISSN 0003-2697
CTAB-GEL
MATERIALS
ELECTROPHORESIS
321
AND METHODS
Chemicals and standard proteins. Analytical-grade sodium acetate and glacial acetic acid were obtained from J. T. Baker Chemical Co. Acrylamide (electrophoresis grade), methylenebisacrylamide, and N,N,N’.N’-tetramethylethylenediamine were from Eastman. Cetyltrimethylammonium bromide (CTAB), Coomassie brilliant blue R-250, and ammonium persulfate were obtained from Sigma. Urea was from Schwarz-Mann, and 2-mercaptoethanol was from Calbiochem. The standard proteins obtained from Sigma were: adolase (rabbit muscle), pyruvate kinase (rabbit skeleton), fructose-6-phosphate kinase (rabbit muscle), y-globulin, ovalbumin, myoglobin, hemoglobin, and lactic dehydrogenase (rabbit muscle). cr-Amylase and carboxypeptidase A were from Worthington. Bovine serum albumin was from Calbiochem. D-Amino acid oxidase (pig kidney) was from Schwarz Bioresearch. Phosphorylase B was from Boeringer Mannheim. Concanavalin A was from Miles Laboratories. Preparation ofprotein Samples. All standard proteins were dissolved at approximately 0.25-0.5 mgiml in 0.01 M sodium acetate, pH 4.6, 0.5% CTAB, and 1.0% 2-mercaptoethanol and were boiled for 5 min or incubated at 37°C for 2 hr. At the end of the boiling or incubation period. solid urea was added to give a final concentration of 6.0 M. After approximately 30 min of further incubation, the protein solutions were ready for loading onto the gel. Preparation of polyacrylamide gels and electrode buffer. Solution A: 30.0 g% acrylamide, 0.45 g% methylene bisacrylamide. Solution B: 245 ml of 0.2 M acetic acid, 355 ml of 0.2 M sodium acetate, 2 g of CTAB, and water to a volume of 1000 ml. Solution C: 2.5 mgiml of ammonium persulfate. For making 10% acrylamide gel in a total of 12 gel tubes (6.0 mm i.d. and 10 cm long), 12 ml of solution A, 16.2 ml of solution B, 1.8 ml of solution C, 6 ml of water, and 0.12 ml of TEMED were thoroughly mixed. The solution was poured into the glass tubes, and water was carefully overlayered to flatten the gel top. All solutions and the above operations were at room temperature. The filled gel tubes were then placed in a 37°C oven, and the polymerization took about 15 min. For 7.5 and 5% acrylamide gels, the preparation was as described above, except the amounts of solution A and water were varied accordingly . The buffer in the electrode compartments was prepared by adding an equal volume of water to solution B. Normally, 2-3 liters were required for electrophoresis in the ISCO Model 419 electrophoresis apparatus. Sample loading and electrophoresis. Twenty microliters of protein solution, prepared as above, was normally loaded onto gels. Azure A dissolved in 0.01 M sodium acetate, pH 4.6, and 10% sucrose (0.5 mgiml)
322
FIG.
1.47%
PANYIM.
THITIPONGPANICH.
1. Electrophoretic pattern cross-linker bisacrylamide.
AND
SUPATIMUSRO
of protein-CTAB complexes in 10% acrylamide Numbers represent the protein code in Table
gel with 1.
was used as a marker dye. Normally, 10 ~1 of the marker dye was electrophoresed for about 3 min prior to sample loading. This was to prevent contact between the Azure A and the protein solution and to sharpen the marker dye boundary. Electrophoresis was performed at constant values of 80- 100 V and S- 10 mA/gel until the Azure A moved close to the bottom of gel tube. The Azure A position was marked by inserting a small wire at the middle of the band. The current tended to increase about 10% at the end of the run. Staining and destaining. The staining solution was that described by Weber and Osborn (2) containing 1.25 g of Coomassie brilliant blue dissolved in 454 ml of 50% methanol and 46 ml of glacial acetic acid. Staining was performed at 80-100°C for 30-60 min. Destaining was done in large volumes of 0.9 N acetic acid at 80-100°C. With frequent changes of 0.9 N acetic acid, all stained bands could be visible in 2-3 hr. Excessively long exposure to hot acetic acid may lead to slow bleaching of stained bands. The gels were stored at room temperature in 0.9 N acetic acid. The mobilities of proteins and Azure A were measured after destain-
CTAB-GEL
TABLE MOLECULAR
WEIGHTS
323
ELECTROPHORESIS
OF PROTEIN
I SUB~NIB
.AND
THEIR
CODES
Subunit molecular weight Code I 2 3 3
Protein
(daltons)
Phosphorylase B Fructose h-phosphate Bovine serum albumin
kinase
Reference
92,500 7x.000 68 .ooo
12 II 7
5 6
Pyruvate kinase Heavy chain y-globulin Ovalbumin
j7.000 50.000 43 .ooo
I2 2 ?
7 x
PIdolase D-Amino
40.000 3 7 .ooo
2 7
?S.OOO 35 .ooo 34.600
2 I? 2
17,000 25,000
I2 I’
17200 15.500
7 2
acid
oxidase
9 IO II
Pepsin Lactic dehydrogenase Carboxypeptidase
I! I3
Concanavalin
13 IS
A
A
u-Amylase Myoglobin Hemoglobin
ing, and relative mobility was obtained protein by that of the dye. RESULTS
by dividing
the mobility
of the
AND DISCUSSION
By converting globular proteins to rod-like structures with approximately equal charge per unit weight, SDS made these proteins separable in SDS-polyacrylamide gel electrophoresis on the basis of size (2,4). CTAB. a cationic detergent, should similarly change a globular protein to one with a rod-like structure and, therefore. should be equally useful in estimating protein molecular weights. Unfortunately, there were many technical problems associated with CTAB-polyacrylamide gel electrophoresis, namely, uniform polymerization of the gel and staining, and destaining procedures. CTAB tended to form precipitates during gel polymerization and, thus, made the gel structure variable. CTAB bound and precipitated the Coomassie blue and had to be removed prior to successful staining. To surmount these problems, we polymerized the acrylamide at 37°C to obtain a uniform gel structure and performed staining and desgianing at 80- 100°C to prevent precipitation of the CTAB complex and to accelerate the tasks. The results, as shown in Fig. 1. demonstrate good resolution of proteins ranging in molecular weight from 17,000 to 68,000 daltons. It is evident that the electrophoretic mobilities are in the order: myoglobin > concanavalin A > lactic dehydrogenase > adolase > ovalbumin > pyruvate kinase > bovine serum albumin. After examining the molecular weights of these protein subunits (Table 1). it
324
PANYIM.
THITIPONGPANICH,
AND
SUPATIMUSRO
lo-
0.3
0.4
0.5
Relative
0.6
0.7
mobility
FIG. 2. A linear relationship between log molecular weights and relative electrophoretic mobilities. The electrophoresis was performed in 10% acrylamide gel with 1.47% crosslinker. Numbers denote the protein code in Table 1.
becomes apparent that their electrophoretic mobilities vary directly with the subunit molecular weights. This is more clearly seen in Fig. 2, in which relative electrophoretic mobilities are plotted against their log molecular weights; a linear relationship between these two parameters was observed. Figure 2 shows the linear relationship within the molecular weight range of 15,000-70,000 daltons, using 10% acrylamide gel with 1.47% crosslinker at pH 4.6. Increasing the amount of cross-linker alone, as seen in
0.2
0.4 Relative
II.6
mobility
FIG. 3. Dependence of electrophoretic mobility on the amount of cross-linker. trophoresis was performed in a constant 10% acrylamide gel with 1.47% (right curve), (middle curve), and 5.67% (left curve) cross-linker.
Elec2.90%
CTAB-GEL
325
ELECTROPHORESIS
FIG. 4. Dependence of electrophoretic mobility on acrylamide phoresis was performed in a constant 1.47% cross-linker with (middle curve), and 10% (left curve) acrylamide.
concentration. Eiectro5% (right curve). 7.5%
the Fig. 3, leads to deviation from the linearity. At 5.6% cross-linker, the linearity extends from 15,000 to about 40,000 daltons. Changing acrylamide concentration at a constantly low cross-linker concentration, as shown in the Fig. 4, does not lead to deviation from linearity. A linear relationship of log molecular weights and relative mobilities is observed in all three acrylamide concentrations. Acrylamide concentration is known to alter lengths of gel fibers per unit volume, while the cross-linker, methylenebisacrylamide, changed the thickness of the gel fibers (5). Apparently, the linear relationship between log molecular I
0
2.5
5.0
7.5 % acrylamide
10.0
FIG. 5. Relationship between log relative mobility and acrylamide concentration constant 1.47% cross-linker. Numbers represent the protein code in Table 1.
in a
326
PANYIM.
THITIPONGPANICH.
4
:’
AND
SUPATIMUSRO
,/:.;
2
L
6
a
10
KrxlOO
FIG. 6. Plot of retardation coefficient (K,) versus molecular weight for seven proteins. The retardation coefficient was computed from the data in Fig. 5.
standard
weight and relative mobility in the CTAB-polyacrylamide gel system is more sensitive to alteration in the thickness than in the length of gel fibers. The results indicate that the structure of the gel pore is a determining factor in the linear relationship of log molecular weight and relative mobility of proteins. Earlier investigators found that CTAB-polyacrylamide gel gives a linear relationship over a short molecular weight range of lO,OOO-20,000 daltons (6). However, optimal conditions (e.g.. cross-linker, acrylamide concentration, or pH) were not explored. When log relative mobility is plotted against acrylamide concentration at a constant cross-linker concentration, as shown in Fig. 5, a series of linear relationships, as predicted by Ferguson’s ‘equation (7), are obtained. Upon extrapolation, all lines intersect approximately at zero acrylamide concentration, indicating that these proteins move at comparable mobilities in free solution (5,8). Therefore, the CTAB-bound proteins should be fractionated in CTAB-polyacrylamide gel electrophoresis solely on basis of size difference (8,9). In SDS-polyacrylamide gel electrophoresis, in which separation was shown to be based on size, SDS-bound proteins were similarly shown to have comparable mobilities in the free solution (9- 11). When molecular weight is plotted against its retardation coefficient, as shown in Fig. 6, a parabolic curve could be best drawn through these points. Frank and Rodbard (11) had demonstrated that the plot of molecular weight versus its retardation coefficient in SDSgel electrophoresis yielded a parabola. Schick (3) had previously shown that N-cetylpyridinium chloride could convert the electrophoretic mobilities of six selected proteins to become linearly related to their molecular weights. Such an electrophoretic system, using 10% acrylamide, 2.70% cross-linker. was claimed to yield a
CTAB-GEL
37-7
ELECTROPHORESIS
linear relationship in a molecular weight range from 17,000 to 170,000 daltons, even without reduction of disulfide linkages. Our present system, under a comparable condition ( 10% acrylamide, 2.90% cross-linker), gives the linear relationship only from 15,000 to 40,000 daltons, or to 70,000 daltons if the cross-linker is reduced to 1.47%. Although our system and Schick’s system differ in the structure of cationic detergents, in pH, and in initiator for gel polymerization, it is difficult to explain the different extent of linearity on these bases. Neville (9) had earlier pointed out the sigmoidal relationship of log molecular weight and relative mobility of the protein-SDS complex and had further demonstrated that a linear relationship could be attenable in the molecular weight range of only 15,00070.000 daltons using comparable acrylamide and cross-linker concentrations. It is noteworthy that our procedure takes 3 hr for electrophoresis, 1 hr for staining, and 2-3 hrs for destaining: therefore, the analysis is not time-consuming and can be accomplished in less than 10 hr. In addition, the procedure is greatly simplified and, consequently, less tedious. As pointed out earlier, CTAB precipitates nucleic acids (13), whereas SDS does not, therefore, the CTAB electrophoresis should allow analysis of proteins in the presence of nucleic acids. For example, an analysis of the total chromosomal proteins of nucleus, without prior removal of DNA, could be envisaged. ACKNOWLEDGMENTS We ceived Health
wish to thank Dr. Bhinyo Panijpan for his valuable financial support from the Faculty of Science, Mahidol Organization.
discussion. University,
This work reand the World
REFERENCES I. Shapiro, A. L., Vinuela. E. and Maizel, J. V.. Jr. (1967) Biochem. Biophy~. Rrs. Commun. 28, 815-820. 2. Weber, K., and Osborn, M. (1970) J. Bid. Chem. 244. 4406-4412. 3. Schick, M. (1975) Anal. Biochem. 63, 345-349. 4. Reynolds, J. A., and Tanford. C. (197O)J. Bid. C17rm. 245, 5161-5165. 5. Chrambach, A., and Rodbard. D. (1971) Science 172, 440-451. 6. Williams, J. G.. and Gratzer. W. B. (1971) J. Chromntogr. 57, 121-125. 7. Ferguson, K. A. (1964) Metabolism 13, 985- 1002. 8. Hedrick, J., and Smith, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. 9. Neville, D. M., Jr. (1971) J. Bid. Chrm. 246, 6338-6334. 10. Weber. K., and Osborn. M. (1975) in The Proteins. Neurath. H.. and Hill. R. L.. eds.). 3rd ed.. Vol. 1, pp. 179-223. Academic Press, New York. Il. Frank, R. N.. and Rodbard. D. (1975) Arch. Biochrm. Biophys. 171, I-13. 12. Klotz, I. M., Darnall, E. W.. and Langerma. N. R. (1975) in The Proteins. (Neurath. H.. and Hill, R. L.. eds.). 3rd ed.. Vol. 1, pp. 293-411, Academic Press, New York. 13. Reitz. M. S., Jr.. Abrell. J. W.. Trainor, C. D.. and Gallo, R. C. (1972) Biothem. Biophys. Res. Commun. 49, 30-38.