ANALYTICAL
BIOCHEMISTRY
88, 644-648
(1978)
Comparison of the Extraction of RNA from Rat Liver by the Diethyl Pyrocarbonate-Sodium Dodecyl Sulfate and Phenol-Chloroform Methods DANIEL
S. H. LIU, GLENN HILTIBRAN,THOMAS
CASTLE, AND
ARLAN RICHARDSON Department
of Chemistry,
Illinois
State
University,
Normal,
Illinois
61761
Received November 16, 1977; accepted March 10, 1978 A higher yield of RNA extracted from rat liver by the diethyl pyrocarbonatesodium dodecyl sulfate method compared to the phenol-chloroform method was consistently obtained. There was no qualitative difference observed in the types of RNA isolated by the two methods. The diethyl pyrocarbonate-sodium dodecyl sulfate technique was shown to be an efficient method for the extraction of poly(A)-containing RNA from rat liver.
Solymosy et al. (1) first described in 1968 a method employing diethyl pyrocarbonate (DEP)-sodium dodecyl sulfate (SDS) to extract RNA from eukaryotic cells. This method is relatively simple and quick and has been used by numerous laboratories for isolating RNA from a variety of organisms (2). Solymosy et al. (3) have described the advantages of the DEP-SDS method in isolating RNA over the conventional methods using phenol. In general, the phenol methods used for RNA isolation are multistep procedures which are affected by ionic strength, pH, and temperature (3). Phenol extractions have also been reported to result in the aggregation and loss of RNA (4,5). In 1970, Solymosy et al. (3) compared the extraction of rRNA from chlorophyll-containing tissues by the DEP-SDS and phenol methods. The results of this study suggested that the DEP-SDS method was superior to the phenol method because higher yields of rRNA were obtained. In 1970, it became possible to isolate mRNA from eukaryotic cells based on the ability of the poly(A) segment on the 3’-end of the mRNA to bind with poly(U) or oligo(dT) (6). To extract mRNA, the phenol method had to be modified because the poly(A) segment became bound to denatured protein at a pH of 7 and was lost (7). By adjusting the phenol pH to 8.3 to 9.0, loss of poly(A)-containing RNA could be minimized (8). The use of phenol-chloroform as described by Aviv and Leder (9) was also found to be satisfactory for isolation of poly(A)-containing mRNA and currently is used in mRNA isolation. Krystosek et al. 0003-2697/78/0882-0644$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
644
EXTRACTION
OF RAT
LIVER
RNA
645
(10) have isolated poly(A)-containing RNA by extracting reticulocyte polyribosomes with 0.5% SDS; however, there have been no reports where the DEP-SDS method of Solymosy et al. (3) has been used to isolate poly(A)-containing RNA. In this study, isolation of RNA from rat liver and the isolation of poly(A)-containing RNA from rat hepatocytes by the DEP-SDS method of Solymosy et al. (3) and the phenol-chloroform method of Aviv and Leder (9) are compared. MATERIALS
AND METHODS
Preparation ofRNA. The liver postmitochondrial supematant (PMS) of 6-month-old female Sprague-Dawley rats was obtained, as described by Richardson et al. (11). Hepatocytes of 6-month-old rats were prepared by a modification of the method of Howard et al. (12), as described previously (13). Radioactively labeled RNA was prepared by incubating hepatocytes (IO7 cells/ml) in Eagle’s medium (14) containing 3.3 &i/ml [3H]orotic acid (23 Ci/mmol) at 37°C for 30 and 60 min. Cells were then washed and collected by low-speed centrifugation. RNA from PMS or hepatocytes was extracted by either the DEP-SDS method of Solymosy et al. (3) or the phenol-chloroform method of Aviv and Leder (9). RNA, DNA, and protein determination. The concentration of RNA extracted from the PMS or hepatocytes was determined, as described by Munro and Fleck (15) using 1.0 A,,, = 32 pg of RNA/ml. The concentrations of protein and DNA in these extracts were determined by the Lowry method (16) and the indole method (17), respectively. Oligo(dT) -cellulose chromatography. The RNA extracted from hepatocytes by the two methods was suspended in a buffer (0.1 M Tris-HCl, pH 7.5, and 1 mg/ml EDTA) and heat denatured at 70°C for 3 min. The samples were brought to 0.5 M KC1 and passed over a 0.25-g column of oligo(dT)-cellulose as described by Aviv and Leder (9). The nonbound fraction was eluted with a buffer containing 0.5 M KC1 and 0.1 M Tris-HCl (pH 7.5). The poly(A)-containing fraction which remained bound to the column was eluted with distilled water. The poly(A)-containing solution was readjusted to 0.5 M KCl, and the solution was rechromatographed on the oligo(dT)-cellulose column a second time to ensure purity. The radioactivity in the different fractions were determined by scintillation counting in NCS-Omnifluor. RESULTS
AND DISCUSSION
The only previous comparison of the DEP-SDS method and a phenol (19) method for extracting RNA was done by Solymosy et al. (3) using chlorophyll-containing tissues. Table 1 compares the isolation of RNA from rat liver PMS or hepatocytes by the DEP-SDS and phenol-chloroform methods. The phenol-chloroform method described by Aviv and
646
LIU
ET AL.
Leder (9) was chosen over the other phenol methods because it is commonly used in the isolation of poly(A)-containing RNA. From Table 1 it is evident that the yield of RNA isolated from liver PMS by the DEPSDS method was always higher (l.Cfold) than that prepared by the phenol-chloroform method. Solymosy et al. (3) found a 1.6-fold higher yield of rRNA by the DEP-SDS method than by a phenol method. The yield of RNA isolated from hepatocytes was greater (1.2-fold) for the DEP-SDS method than for the phenol-chloroform method. These results also indicate the yield of extractable RNA obtained by the two methods from the starting material; however, the DEP-SDS method consistently gave higher yields of RNA than the phenol-chloroform method. In all cases, the RNA extracted from either PMS or hepatocytes by the two methods had a similar contamination of protein, approximately 63 &mg of RNA. There was little DNA contamination in the RNA isolated from liver PMS preparations. The DNA contaminations of RNA isolated from hepatocytes was in the range of 170 to 210 pg/mg of RNA for both methods. The yield of RNA extracted from hepatocytes by the DEP-SDS method was approximately 60% whereas by the phenol-chloroform method the yield was only 29%. Solymosy et al. (3) reported that the yield of rRNA from chlorophyll-containing tissues was 85 and 51% for the DEP-SDS and phenol (19) methods, respectively. Because there was a significant difference in the amount of RNA obtained from liver by the two methods, it was of interest to determine if there TABLE EXTRACTION
OF RNA
FROM
LIVER
DEP-SDS I. Total RNA extracted*: PMS Hepatocytes II.
Poly(A) RNA extractedc: 30-min incubation 60-min incubation
1
method
1560 k 168 377 k 22
21.8% 11.1%
PMS
AND HEPATOCYTES Phenol-chloroform method
Ratio”
1175 2 187 167 2 17
1.4 2.3
17.8% 12.5%
-
a The ratio of RNA extracted by the DEP-SDS method compared to the phenol-chloroform method is given. b The concentration of RNA isolated from rat liver PMS is expressed as micrograms of RNA per milliliter of PMS. The concentration of RNA isolated from hepatocytes is expressed as micrograms of RNA extracted per 10’ cells. Each value represents the mean f SEM of three separate experiments. C Radioactively labeled RNA was isolated from hepatocytes by the two methods and subjected to oligo(dT)-cellulose chromatography as described under Materials and Methods. The percentage of radioactivity which bound to the oligo(dT)-cellulose column is given.
EXTRACTION
OF RAT
LIVER
RNA
647
were any qualitative differences in the types of RNA isolated. Figure 1 shows the distribution of RNA obtained from liver PMS by the two methods as determined by polyacrylamide gel electrophoresis. In both cases, the ratio of 28s rRNA to 18s rRNA was approximately 2, indicating little degradation of RNA occurred with either technique during the isolation of RNA. Comparison of the size distribution of the RNA obtained by the two methods with gel electrophoresis indicates that there are no qualitative differences in the types of RNA isolated. Similar results were obtained for the extracts of RNA prepared from hepatocytes. Next, the isolation of poly(A)-containing RNA by the two methods was compared. Because mRNA makes up a very small fraction of the total RNA in a cell (20), the amount of radioactively-labeled RNA bound to oligo(dT)-cellulose was used to compare the extraction of poly(A)containing RNA by the two methods. In hepatocytes incubated with [3Hjorotic acid for 30 or 60 min, 18 to 22% or 11 to 13% of the radisactively labeled RNA bound to oligo(dT)-cellulose, respectively. Only 0.5 to 1.5% of the total RNA bound to oligo(dT)-cellulose. The relatively high percentage of radioactively labeled poly(A)-containing
I
28s
FIG. 1. SDS-polyacrylamide gel electrophoresis of RNA isolated from PMS polyacrylamide gels (2.7%) were prepared, as described by Weber and Osbom (18). RNA (30 pg) extracted from the rat liver PMS by either the phenol-chloroform method (A) or the DEP-SDS method (B) was run on polyacrylamide gels at room temperature for 4 hr at 8 mA/gel, and gels were then scanned at 260 nm. The peaks of 4. 18, and 28s migrating RNA were determined by using yeast tRNA and rat liver rRNA (18 and 28s) as standards.
648
LIU
ET
AL.
RNA probably reflects the preferential synthesis of mRNA during the relatively short periods of labeling. Although the percentage of poly(A)containing RNA isolated by the two methods was similar, the total amount of poly(A)-containing RNA isolated by the DEP-SDS method was twofold greater than by the phenol-chloroform method, because the yield of RNA by the DEP-SDS method was greater (Table 1). Although the DEP-SDS method of Solymosy er al. (3) appears advantageous for the extraction of poly(A)-containing RNA as an analytical sample, the biological activity of the poly(A)-containing RNA by the two methods was not determined. The DEP-SDS method might not be an adequate extraction procedure for preparing functional mRNA, since Ehrenberg et al. (2) reported that DEP reacts with single-stranded RNA, especially the adenylate residues, with subsequent loss of biological activity. Ehrenberg et al. (2) proposed that this problem would be overcome, however, and a comparison of the biological activity of poly(A)-containing ,RNA from both extraction methods is required to resolve this question. ACKNOWLEDGMENTS This study was supported in part by NIH Grant 5 R01 AGOO344-02.
REFERENCES 1. Solymosy, F., Fedorcsak, I., Gulyas, A., Farkas, G. L., and Ehrenberg, L. (1968) Eur. J. Biochem. 5, 520-527. 2. Ehrenberg, L., Fedorcsak, I., and Solymosy, F. (1976) Prog. Nucleic Acid Res. Mol. Biol. 16, 189-262. 3. Solymosy, F., Lazar, G., and Bagi, G. (1970) Anal. Biochem. 38, 40-45. 4. Maroun, L. E., Driscoll, B. F., and Nordonne, R. M. (1971) Nature New Biol. 231, 270-271. 5. Brawerman, G., Mendecki, J., and Lee, S. Y. (1972) Biochemistry 11, 637-642. 6. Lim, L., and Conellakis, E. S. (1970) Nature (London) 227, 710-712. 7. Cranston, J., Malathi, V. G., and Silber, R. (1973) Fed. Proc. 32, 498. 8. Yogo, Y., and Wimmer, E. (1973) Nature New Biol. 242, 171-174. 9. Aviv, H., and Leder, P. (1972) Proc. Nat. Acad. Sci. USA 69, 1408-1412. 10. Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084. 11. Richardson, A., McGown, E., Henderson, L. M., and Swan, P. B. (1971) Biochim. Biophys. Acta 254, 468-477. 12. Howard, R. R., Lee, J. C., and Pesch, L. A. (1973)J. Cell Biol. 57, 642-658. 13. Ricca, G. A., Liu, D. S. H., Coniglio, J. J., and Richardson, A. (1978)5. Cell Physiol. (in press). 14. Eagle, H. (1959) Science 130, 432-437. 15. Munro, H. N., and Fleck, A. (1966) Analyst 91, 78-88. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, F. J. (1951) J. Biol. Chem. 193, 265-272.
17. Ceriotti, G. (1952) J. Biol. Chem. 198, 297-303. 18. Weber, K. and Osbom, M. (1975) in The Proteins (Neurath, H., and Hill, R. L., eds.), 3rd ed., Vol. 1, pp. 179-223, Academic Press, New York. 19. Ingle, J., and Bums, R. G. (1968)Biochem. J. 110, 605-606. 20. Singer, M. F.. and Leder, P. (1966) Annu. Rev. Biochem. 35, 195-230.
BIOCHEMISTRY
88, 644-648
(1978)
Comparison of the Extraction of RNA from Rat Liver by the Diethyl Pyrocarbonate-Sodium Dodecyl Sulfate and Phenol-Chloroform Methods DANIEL
S. H. LIU, GLENN HILTIBRAN,THOMAS
CASTLE, AND
ARLAN RICHARDSON Department
of Chemistry,
Illinois
State
University,
Normal,
Illinois
61761
Received November 16, 1977; accepted March 10, 1978 A higher yield of RNA extracted from rat liver by the diethyl pyrocarbonatesodium dodecyl sulfate method compared to the phenol-chloroform method was consistently obtained. There was no qualitative difference observed in the types of RNA isolated by the two methods. The diethyl pyrocarbonate-sodium dodecyl sulfate technique was shown to be an efficient method for the extraction of poly(A)-containing RNA from rat liver.
Solymosy et al. (1) first described in 1968 a method employing diethyl pyrocarbonate (DEP)-sodium dodecyl sulfate (SDS) to extract RNA from eukaryotic cells. This method is relatively simple and quick and has been used by numerous laboratories for isolating RNA from a variety of organisms (2). Solymosy et al. (3) have described the advantages of the DEP-SDS method in isolating RNA over the conventional methods using phenol. In general, the phenol methods used for RNA isolation are multistep procedures which are affected by ionic strength, pH, and temperature (3). Phenol extractions have also been reported to result in the aggregation and loss of RNA (4,5). In 1970, Solymosy et al. (3) compared the extraction of rRNA from chlorophyll-containing tissues by the DEP-SDS and phenol methods. The results of this study suggested that the DEP-SDS method was superior to the phenol method because higher yields of rRNA were obtained. In 1970, it became possible to isolate mRNA from eukaryotic cells based on the ability of the poly(A) segment on the 3’-end of the mRNA to bind with poly(U) or oligo(dT) (6). To extract mRNA, the phenol method had to be modified because the poly(A) segment became bound to denatured protein at a pH of 7 and was lost (7). By adjusting the phenol pH to 8.3 to 9.0, loss of poly(A)-containing RNA could be minimized (8). The use of phenol-chloroform as described by Aviv and Leder (9) was also found to be satisfactory for isolation of poly(A)-containing mRNA and currently is used in mRNA isolation. Krystosek et al. 0003-2697/78/0882-0644$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
644
EXTRACTION
OF RAT
LIVER
RNA
645
(10) have isolated poly(A)-containing RNA by extracting reticulocyte polyribosomes with 0.5% SDS; however, there have been no reports where the DEP-SDS method of Solymosy et al. (3) has been used to isolate poly(A)-containing RNA. In this study, isolation of RNA from rat liver and the isolation of poly(A)-containing RNA from rat hepatocytes by the DEP-SDS method of Solymosy et al. (3) and the phenol-chloroform method of Aviv and Leder (9) are compared. MATERIALS
AND METHODS
Preparation ofRNA. The liver postmitochondrial supematant (PMS) of 6-month-old female Sprague-Dawley rats was obtained, as described by Richardson et al. (11). Hepatocytes of 6-month-old rats were prepared by a modification of the method of Howard et al. (12), as described previously (13). Radioactively labeled RNA was prepared by incubating hepatocytes (IO7 cells/ml) in Eagle’s medium (14) containing 3.3 &i/ml [3H]orotic acid (23 Ci/mmol) at 37°C for 30 and 60 min. Cells were then washed and collected by low-speed centrifugation. RNA from PMS or hepatocytes was extracted by either the DEP-SDS method of Solymosy et al. (3) or the phenol-chloroform method of Aviv and Leder (9). RNA, DNA, and protein determination. The concentration of RNA extracted from the PMS or hepatocytes was determined, as described by Munro and Fleck (15) using 1.0 A,,, = 32 pg of RNA/ml. The concentrations of protein and DNA in these extracts were determined by the Lowry method (16) and the indole method (17), respectively. Oligo(dT) -cellulose chromatography. The RNA extracted from hepatocytes by the two methods was suspended in a buffer (0.1 M Tris-HCl, pH 7.5, and 1 mg/ml EDTA) and heat denatured at 70°C for 3 min. The samples were brought to 0.5 M KC1 and passed over a 0.25-g column of oligo(dT)-cellulose as described by Aviv and Leder (9). The nonbound fraction was eluted with a buffer containing 0.5 M KC1 and 0.1 M Tris-HCl (pH 7.5). The poly(A)-containing fraction which remained bound to the column was eluted with distilled water. The poly(A)-containing solution was readjusted to 0.5 M KCl, and the solution was rechromatographed on the oligo(dT)-cellulose column a second time to ensure purity. The radioactivity in the different fractions were determined by scintillation counting in NCS-Omnifluor. RESULTS
AND DISCUSSION
The only previous comparison of the DEP-SDS method and a phenol (19) method for extracting RNA was done by Solymosy et al. (3) using chlorophyll-containing tissues. Table 1 compares the isolation of RNA from rat liver PMS or hepatocytes by the DEP-SDS and phenol-chloroform methods. The phenol-chloroform method described by Aviv and
646
LIU
ET AL.
Leder (9) was chosen over the other phenol methods because it is commonly used in the isolation of poly(A)-containing RNA. From Table 1 it is evident that the yield of RNA isolated from liver PMS by the DEPSDS method was always higher (l.Cfold) than that prepared by the phenol-chloroform method. Solymosy et al. (3) found a 1.6-fold higher yield of rRNA by the DEP-SDS method than by a phenol method. The yield of RNA isolated from hepatocytes was greater (1.2-fold) for the DEP-SDS method than for the phenol-chloroform method. These results also indicate the yield of extractable RNA obtained by the two methods from the starting material; however, the DEP-SDS method consistently gave higher yields of RNA than the phenol-chloroform method. In all cases, the RNA extracted from either PMS or hepatocytes by the two methods had a similar contamination of protein, approximately 63 &mg of RNA. There was little DNA contamination in the RNA isolated from liver PMS preparations. The DNA contaminations of RNA isolated from hepatocytes was in the range of 170 to 210 pg/mg of RNA for both methods. The yield of RNA extracted from hepatocytes by the DEP-SDS method was approximately 60% whereas by the phenol-chloroform method the yield was only 29%. Solymosy et al. (3) reported that the yield of rRNA from chlorophyll-containing tissues was 85 and 51% for the DEP-SDS and phenol (19) methods, respectively. Because there was a significant difference in the amount of RNA obtained from liver by the two methods, it was of interest to determine if there TABLE EXTRACTION
OF RNA
FROM
LIVER
DEP-SDS I. Total RNA extracted*: PMS Hepatocytes II.
Poly(A) RNA extractedc: 30-min incubation 60-min incubation
1
method
1560 k 168 377 k 22
21.8% 11.1%
PMS
AND HEPATOCYTES Phenol-chloroform method
Ratio”
1175 2 187 167 2 17
1.4 2.3
17.8% 12.5%
-
a The ratio of RNA extracted by the DEP-SDS method compared to the phenol-chloroform method is given. b The concentration of RNA isolated from rat liver PMS is expressed as micrograms of RNA per milliliter of PMS. The concentration of RNA isolated from hepatocytes is expressed as micrograms of RNA extracted per 10’ cells. Each value represents the mean f SEM of three separate experiments. C Radioactively labeled RNA was isolated from hepatocytes by the two methods and subjected to oligo(dT)-cellulose chromatography as described under Materials and Methods. The percentage of radioactivity which bound to the oligo(dT)-cellulose column is given.
EXTRACTION
OF RAT
LIVER
RNA
647
were any qualitative differences in the types of RNA isolated. Figure 1 shows the distribution of RNA obtained from liver PMS by the two methods as determined by polyacrylamide gel electrophoresis. In both cases, the ratio of 28s rRNA to 18s rRNA was approximately 2, indicating little degradation of RNA occurred with either technique during the isolation of RNA. Comparison of the size distribution of the RNA obtained by the two methods with gel electrophoresis indicates that there are no qualitative differences in the types of RNA isolated. Similar results were obtained for the extracts of RNA prepared from hepatocytes. Next, the isolation of poly(A)-containing RNA by the two methods was compared. Because mRNA makes up a very small fraction of the total RNA in a cell (20), the amount of radioactively-labeled RNA bound to oligo(dT)-cellulose was used to compare the extraction of poly(A)containing RNA by the two methods. In hepatocytes incubated with [3Hjorotic acid for 30 or 60 min, 18 to 22% or 11 to 13% of the radisactively labeled RNA bound to oligo(dT)-cellulose, respectively. Only 0.5 to 1.5% of the total RNA bound to oligo(dT)-cellulose. The relatively high percentage of radioactively labeled poly(A)-containing
I
28s
FIG. 1. SDS-polyacrylamide gel electrophoresis of RNA isolated from PMS polyacrylamide gels (2.7%) were prepared, as described by Weber and Osbom (18). RNA (30 pg) extracted from the rat liver PMS by either the phenol-chloroform method (A) or the DEP-SDS method (B) was run on polyacrylamide gels at room temperature for 4 hr at 8 mA/gel, and gels were then scanned at 260 nm. The peaks of 4. 18, and 28s migrating RNA were determined by using yeast tRNA and rat liver rRNA (18 and 28s) as standards.
648
LIU
ET
AL.
RNA probably reflects the preferential synthesis of mRNA during the relatively short periods of labeling. Although the percentage of poly(A)containing RNA isolated by the two methods was similar, the total amount of poly(A)-containing RNA isolated by the DEP-SDS method was twofold greater than by the phenol-chloroform method, because the yield of RNA by the DEP-SDS method was greater (Table 1). Although the DEP-SDS method of Solymosy er al. (3) appears advantageous for the extraction of poly(A)-containing RNA as an analytical sample, the biological activity of the poly(A)-containing RNA by the two methods was not determined. The DEP-SDS method might not be an adequate extraction procedure for preparing functional mRNA, since Ehrenberg et al. (2) reported that DEP reacts with single-stranded RNA, especially the adenylate residues, with subsequent loss of biological activity. Ehrenberg et al. (2) proposed that this problem would be overcome, however, and a comparison of the biological activity of poly(A)-containing ,RNA from both extraction methods is required to resolve this question. ACKNOWLEDGMENTS This study was supported in part by NIH Grant 5 R01 AGOO344-02.
REFERENCES 1. Solymosy, F., Fedorcsak, I., Gulyas, A., Farkas, G. L., and Ehrenberg, L. (1968) Eur. J. Biochem. 5, 520-527. 2. Ehrenberg, L., Fedorcsak, I., and Solymosy, F. (1976) Prog. Nucleic Acid Res. Mol. Biol. 16, 189-262. 3. Solymosy, F., Lazar, G., and Bagi, G. (1970) Anal. Biochem. 38, 40-45. 4. Maroun, L. E., Driscoll, B. F., and Nordonne, R. M. (1971) Nature New Biol. 231, 270-271. 5. Brawerman, G., Mendecki, J., and Lee, S. Y. (1972) Biochemistry 11, 637-642. 6. Lim, L., and Conellakis, E. S. (1970) Nature (London) 227, 710-712. 7. Cranston, J., Malathi, V. G., and Silber, R. (1973) Fed. Proc. 32, 498. 8. Yogo, Y., and Wimmer, E. (1973) Nature New Biol. 242, 171-174. 9. Aviv, H., and Leder, P. (1972) Proc. Nat. Acad. Sci. USA 69, 1408-1412. 10. Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084. 11. Richardson, A., McGown, E., Henderson, L. M., and Swan, P. B. (1971) Biochim. Biophys. Acta 254, 468-477. 12. Howard, R. R., Lee, J. C., and Pesch, L. A. (1973)J. Cell Biol. 57, 642-658. 13. Ricca, G. A., Liu, D. S. H., Coniglio, J. J., and Richardson, A. (1978)5. Cell Physiol. (in press). 14. Eagle, H. (1959) Science 130, 432-437. 15. Munro, H. N., and Fleck, A. (1966) Analyst 91, 78-88. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, F. J. (1951) J. Biol. Chem. 193, 265-272.
17. Ceriotti, G. (1952) J. Biol. Chem. 198, 297-303. 18. Weber, K. and Osbom, M. (1975) in The Proteins (Neurath, H., and Hill, R. L., eds.), 3rd ed., Vol. 1, pp. 179-223, Academic Press, New York. 19. Ingle, J., and Bums, R. G. (1968)Biochem. J. 110, 605-606. 20. Singer, M. F.. and Leder, P. (1966) Annu. Rev. Biochem. 35, 195-230.
