ANALYTICAL
66, 64-68 (1975)
BIOCHEMISTRY
The Differential and
Proteins
Precipitation from
Aqueous
of Nucleic Solutions
Acids
by Ethanol
J. WILCOCKSON Fachbereich Biologie-Botanik, Phil&w-Universitat, 355 MarburglLahn, West Germany Received May 20, 1974; revised October 21, 1974 The addition of an appropriate mixture of ethanol, water and sodium perchlorate to crude extracts of some biological material results in the selective precipitation of nucleic acids. Detergent extracts of tissue-cultured plant cells treated with this reagent yielded as much as 95-100% of the nucleic acid while a similar percentage of the total protein remained in solution.
Sodium perchlorate (NaC104 * HzO) in high concentration will remove from solution the detergent sodium dodecyl sulfate and protein complexed with it (1). RNA and DNA remain behind in solution although the yield of the latter is much reduced if present as very large molecules. This method was found to be effective in the preparation of plant viral RNAs and in most cases significant increases in yield and speed of preparation of infectious RNAs have been achieved (2). It was noted that even if all protein was not removed with the salted out detergent, that which remained in solution was not precipitated on addition of 2 volumes of ethanol to the solution. This paper describes a reduction of the procedure to one step whereby the final desired concentrations of both perchlorate and ethanol are achieved simultaneously. It was also the intention to eliminate the stage where detergent was removed from solution since this appeared to be the source of loss of some DNAs. METHODS
AND
MATERIALS
Soybean cells (Glycine max L. Merr.) of a strain originating from the laboratory of Dr. Gamborg were grown in suspension culture in the medium of Miller (3). Erlenmeyer flasks of 300 ml and containing about 125 ml of culture were incubated at 28” with gyrorotary shaking of 120 strokes/min in the dark. Three cultures were labeled either with [methyl-3H] thymidine, approximately 0.1 &i/ml and 56 Cilmmol (A), or D-3 [ 14C]phenylalanine universally labeled, approximately 0.025 &i/ml and 477 mCi/mmol (B), or with both isotopic reagents (C). Radioactive 64 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
DIFFERENTIAL
PRJXIPITATION
BY
ETHANOL
65
materials were obtained from The Radiochemical Centre, Amersham, Bucks., U.K. via Amersham Buchler, Frankfurt, West Germany. Both labels were added to early log phase cultures about 60 h before the cells were processed as follows. Cell suspensions were poured through fine nylon gauze (about 10 pm pores) and washed profusely with water. Acid-washed sand equal to about half the wet weight of the washed cell suspension was added to it in a cold mortar together with enough cold saline-EDTA (4) to make a thin paste for grinding. After grinding to disrupt the cells more salineEDTA was added and the contents of the mortar transferred to a centrifuge tube. Sodium dodecyl sulfate (25%) was added to give a final concentration of about 5% and the tube heated to 50-60” for a few minutes and mixed well with a glass stirrer. Cell debris and sand were pelleted by a few minutes centrifugation at low speed. It was arranged that the volume of the supernatant should exceed the volume of the pellet by a factor of about five. Six volumes of either 80% ethanol or the ethanolic perchlorate reagent (EPR) were pipetted vigorously into aliquots of the supematants to give rapid mixing. Consisting of about 80% v/v ethanol and 30-35% w/v perchlorate, EPR was prepared by mixing together a saturated solution of the salt in water with four volumes of a saturated solution in ethanol (approximately 135% and 10% w/v respectively). A few crystals may settle out of the solution on standing but since the relevant properties of the reagent do not appear to be critically concentration dependent precise figures were not determined. After 15-20 min at 10” precipitated material in the tubes was pelleted by low speed centrifugation for about 10 min. The precipitates were all washed in cold 70% ethanol, again centrifuged and after allowing to drain, were dissolved in dilute saline sodium citrate (0.1 x SSC) (4). It is important to wash away perchlorate from the precipitates; failure to do this will render the pellets difficult to break up and dissolve. Generally the same volume of 0.1 X SSC was used as the volume of the aliquot of cell extract treated. Small samples from these solutions were taken and pipetted on to Whatman GFB glass fiber disks (2.5 cm) for determination of radioactivity. The solutions were made up to 2.5% sodium dodecyl sulfate by addition of a 25% solution and again heated like the initial extract. Eighty percent ethanol or EPR was added and a second precipitate derived and sampled for radioactivity. The whole process was again repeated to give a third and final pellet which was dissolved, sampled and also counted for radioactivity. After drying, each glass fiber disk was placed in the bottom of a polyethylene scintillation bottle and 2 ml of scintillator fluid added (4 g
66
J. WILCOCKSON
2,5diphenyloxazole and 0.25 g 1,4 bis(5-phenyloxazol-2-yl) benzene/liter of toluene). Radioactivity was determined in a Packard TriCarb Liquid Scintillation Spectrometer, model 3380. RESULTS
Some immediate indication of the different effects produced by the two agents of precipitation is given. A more dense flocculence is produced by the ethanol than by EPR and after centrifuging the size of the pellet is greater with the former reagent. However it can be seen from Table 1. that the loss of material from the EPR precipitated samples is not generalized but confined almost entirely to the protein fraction of the ethanol precipitates. While both ethanol and EPR precipitates are, after washing in 70% ethanol, easily dispersed only the latter gives a clear solution. Ethanol precipitates are alway very turbid presumably because of colloidal denatured protein. Successive precipitations with either reagent result in small progressive losses of both DNA (3H) and of protein (‘“C). However EPR fails even at the first precipitation to bring down about 95% of the protein compared with ethanol. At the same time EPR is almost as efficient as ethanol at precipitating DNA at any of the stages. In the doubly labeled culture (Table 2), it is obvious that despite these small progressive losses with repeated precipitations there is also a progressive improvement in the ratio of DNA to protein with EPR and none with the ethanol.
TABLE 1 PRECIPITATIONOF DNA ANDPROTEINFROMEXTRACTSOFSOYBEANCELL CULTURESLABELEDWITH 3HTdR ANDY-LABELEDAMINOACID Counts/min/ml of dissolved precipitate precipitating agent!
PPt.
80% ethanol a
EPR
label
b
b/a x 100
A
3H
first second third
3465 3315 3151
3343 3263 3061
96.5 98.4 97.1
B
IT
first second third
6813 4911 4555
298 198 171
4.4 4.0 3.8
culture
a Counts/min/ml of the dissolved precipitate produced respectively by the addition of 6 volumes of 80% ethanol or of EPR to 1 volume of extract.
DIFFERENTIAL
PRECIPITATION
RATIOS OF 3H(DNA) LABEL TO Y EXTRACT OF A DOUBLY LABELED
BY
67
ETHANOL
TABLE 2 (PROTEIN) LABEL IN PRECIPITATES FROM AN CULTURE OF SOYBEAN CELL SUSPENSION Precipitating
Culture
C
Label
3H + ‘Y
agenP
Precipitate
80% ethanol
EPR
first
0.805 0.700 0.830
12.4 14.3 22.3
second third fourthb
1.7
n Ratio
3H counts/min in sample r4C counts/min in sample * The dissolved third precipitate produced by addition of 80% ethanol precipitated for a fourth time by 6 volumes of EPR. Recovery of counts compared with the first ethanol precipitate was about 50% for 3H and about 25% for 14C.
DISCUSSION
It is highly probable that the radioactivity in the first ethanol precipitate represents virtually the total of that present in the two classes of macromolecules since it was not possible to render any more counts insoluble with other denaturing and precipitating agents including trichloracetic acid. Thus it can be said that EPR will, in one step, free almost all the nucleic acid from almost all the protein in a crude extract. Ethanol precipitation is part of most procedures for the preparation of nucleic acids from viruses, bacteria and cells and tissues of higher organisms. The ethanol step usually follows a deproteinization procedure. It is common experience that nucleic acids coprecipitated with proteins are difficult to recover pure again. Indeed if an ethanol precipitated pellet is solubilized and treated with detergent followed by EPR an improvement in the ratio of isotopes in favour of the DNA is obtained (Table 2) but not to the same extent as in one precipitation by EPR alone. It should also be noted that while about 75% of the protein was lost from this EPR treated ethanol precipitate only about half of the radioactivity in the DNA was recovered. Precipitation of nucleic acids by ethanol is most commonly achieved by the addition of 2 or sometimes 2.5 volumes of the reagent. Higher concentrations may precipitate low molecular weight materials and even buffer salts. Ethanolic perchlorate reagent was formulated by trial and error such that on addition to an extract the final concentration of ethanol would be in the range 66-70% and the final perchlorate 25-30% (w/v) and also to keep at a minimum the volume it is necessary to add. It is suggested that since the efficiency of preparation of nucleic acid
68
J. WILCOCKSON
from tissues by this method is only limited by the efficiency of the extraction procedure itself and hardly at all by the EPR step that it may be useful in the chemical estimation of nucleic acids to free a crude extract of interfering substances prior to a colour test. ACKNOWLEDGMENTS This work was completed with the financial support of Deutsche Forschungsgemeinschaft (SFB 103). The advice, encouragement and provision of laboratory facilities by Professor Dietrich Werner of this University is also gratefully acknowledged.
REFERENCES 1. 2. 3. 4.
Wilcockson, J. (1973) Biochem. J. 135, 559-561. Wilcockson, J., and Hull, R. (1974) J. Gen. Viral. 23, 107-l 11. Miller, C. 0. (1967)Ann. N.Y. Acad. Sci. 144, 2.51-257. Marmur, J. (1961) J. Mol. Biol. 3, 208-218.
66, 64-68 (1975)
BIOCHEMISTRY
The Differential and
Proteins
Precipitation from
Aqueous
of Nucleic Solutions
Acids
by Ethanol
J. WILCOCKSON Fachbereich Biologie-Botanik, Phil&w-Universitat, 355 MarburglLahn, West Germany Received May 20, 1974; revised October 21, 1974 The addition of an appropriate mixture of ethanol, water and sodium perchlorate to crude extracts of some biological material results in the selective precipitation of nucleic acids. Detergent extracts of tissue-cultured plant cells treated with this reagent yielded as much as 95-100% of the nucleic acid while a similar percentage of the total protein remained in solution.
Sodium perchlorate (NaC104 * HzO) in high concentration will remove from solution the detergent sodium dodecyl sulfate and protein complexed with it (1). RNA and DNA remain behind in solution although the yield of the latter is much reduced if present as very large molecules. This method was found to be effective in the preparation of plant viral RNAs and in most cases significant increases in yield and speed of preparation of infectious RNAs have been achieved (2). It was noted that even if all protein was not removed with the salted out detergent, that which remained in solution was not precipitated on addition of 2 volumes of ethanol to the solution. This paper describes a reduction of the procedure to one step whereby the final desired concentrations of both perchlorate and ethanol are achieved simultaneously. It was also the intention to eliminate the stage where detergent was removed from solution since this appeared to be the source of loss of some DNAs. METHODS
AND
MATERIALS
Soybean cells (Glycine max L. Merr.) of a strain originating from the laboratory of Dr. Gamborg were grown in suspension culture in the medium of Miller (3). Erlenmeyer flasks of 300 ml and containing about 125 ml of culture were incubated at 28” with gyrorotary shaking of 120 strokes/min in the dark. Three cultures were labeled either with [methyl-3H] thymidine, approximately 0.1 &i/ml and 56 Cilmmol (A), or D-3 [ 14C]phenylalanine universally labeled, approximately 0.025 &i/ml and 477 mCi/mmol (B), or with both isotopic reagents (C). Radioactive 64 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
DIFFERENTIAL
PRJXIPITATION
BY
ETHANOL
65
materials were obtained from The Radiochemical Centre, Amersham, Bucks., U.K. via Amersham Buchler, Frankfurt, West Germany. Both labels were added to early log phase cultures about 60 h before the cells were processed as follows. Cell suspensions were poured through fine nylon gauze (about 10 pm pores) and washed profusely with water. Acid-washed sand equal to about half the wet weight of the washed cell suspension was added to it in a cold mortar together with enough cold saline-EDTA (4) to make a thin paste for grinding. After grinding to disrupt the cells more salineEDTA was added and the contents of the mortar transferred to a centrifuge tube. Sodium dodecyl sulfate (25%) was added to give a final concentration of about 5% and the tube heated to 50-60” for a few minutes and mixed well with a glass stirrer. Cell debris and sand were pelleted by a few minutes centrifugation at low speed. It was arranged that the volume of the supernatant should exceed the volume of the pellet by a factor of about five. Six volumes of either 80% ethanol or the ethanolic perchlorate reagent (EPR) were pipetted vigorously into aliquots of the supematants to give rapid mixing. Consisting of about 80% v/v ethanol and 30-35% w/v perchlorate, EPR was prepared by mixing together a saturated solution of the salt in water with four volumes of a saturated solution in ethanol (approximately 135% and 10% w/v respectively). A few crystals may settle out of the solution on standing but since the relevant properties of the reagent do not appear to be critically concentration dependent precise figures were not determined. After 15-20 min at 10” precipitated material in the tubes was pelleted by low speed centrifugation for about 10 min. The precipitates were all washed in cold 70% ethanol, again centrifuged and after allowing to drain, were dissolved in dilute saline sodium citrate (0.1 x SSC) (4). It is important to wash away perchlorate from the precipitates; failure to do this will render the pellets difficult to break up and dissolve. Generally the same volume of 0.1 X SSC was used as the volume of the aliquot of cell extract treated. Small samples from these solutions were taken and pipetted on to Whatman GFB glass fiber disks (2.5 cm) for determination of radioactivity. The solutions were made up to 2.5% sodium dodecyl sulfate by addition of a 25% solution and again heated like the initial extract. Eighty percent ethanol or EPR was added and a second precipitate derived and sampled for radioactivity. The whole process was again repeated to give a third and final pellet which was dissolved, sampled and also counted for radioactivity. After drying, each glass fiber disk was placed in the bottom of a polyethylene scintillation bottle and 2 ml of scintillator fluid added (4 g
66
J. WILCOCKSON
2,5diphenyloxazole and 0.25 g 1,4 bis(5-phenyloxazol-2-yl) benzene/liter of toluene). Radioactivity was determined in a Packard TriCarb Liquid Scintillation Spectrometer, model 3380. RESULTS
Some immediate indication of the different effects produced by the two agents of precipitation is given. A more dense flocculence is produced by the ethanol than by EPR and after centrifuging the size of the pellet is greater with the former reagent. However it can be seen from Table 1. that the loss of material from the EPR precipitated samples is not generalized but confined almost entirely to the protein fraction of the ethanol precipitates. While both ethanol and EPR precipitates are, after washing in 70% ethanol, easily dispersed only the latter gives a clear solution. Ethanol precipitates are alway very turbid presumably because of colloidal denatured protein. Successive precipitations with either reagent result in small progressive losses of both DNA (3H) and of protein (‘“C). However EPR fails even at the first precipitation to bring down about 95% of the protein compared with ethanol. At the same time EPR is almost as efficient as ethanol at precipitating DNA at any of the stages. In the doubly labeled culture (Table 2), it is obvious that despite these small progressive losses with repeated precipitations there is also a progressive improvement in the ratio of DNA to protein with EPR and none with the ethanol.
TABLE 1 PRECIPITATIONOF DNA ANDPROTEINFROMEXTRACTSOFSOYBEANCELL CULTURESLABELEDWITH 3HTdR ANDY-LABELEDAMINOACID Counts/min/ml of dissolved precipitate precipitating agent!
PPt.
80% ethanol a
EPR
label
b
b/a x 100
A
3H
first second third
3465 3315 3151
3343 3263 3061
96.5 98.4 97.1
B
IT
first second third
6813 4911 4555
298 198 171
4.4 4.0 3.8
culture
a Counts/min/ml of the dissolved precipitate produced respectively by the addition of 6 volumes of 80% ethanol or of EPR to 1 volume of extract.
DIFFERENTIAL
PRECIPITATION
RATIOS OF 3H(DNA) LABEL TO Y EXTRACT OF A DOUBLY LABELED
BY
67
ETHANOL
TABLE 2 (PROTEIN) LABEL IN PRECIPITATES FROM AN CULTURE OF SOYBEAN CELL SUSPENSION Precipitating
Culture
C
Label
3H + ‘Y
agenP
Precipitate
80% ethanol
EPR
first
0.805 0.700 0.830
12.4 14.3 22.3
second third fourthb
1.7
n Ratio
3H counts/min in sample r4C counts/min in sample * The dissolved third precipitate produced by addition of 80% ethanol precipitated for a fourth time by 6 volumes of EPR. Recovery of counts compared with the first ethanol precipitate was about 50% for 3H and about 25% for 14C.
DISCUSSION
It is highly probable that the radioactivity in the first ethanol precipitate represents virtually the total of that present in the two classes of macromolecules since it was not possible to render any more counts insoluble with other denaturing and precipitating agents including trichloracetic acid. Thus it can be said that EPR will, in one step, free almost all the nucleic acid from almost all the protein in a crude extract. Ethanol precipitation is part of most procedures for the preparation of nucleic acids from viruses, bacteria and cells and tissues of higher organisms. The ethanol step usually follows a deproteinization procedure. It is common experience that nucleic acids coprecipitated with proteins are difficult to recover pure again. Indeed if an ethanol precipitated pellet is solubilized and treated with detergent followed by EPR an improvement in the ratio of isotopes in favour of the DNA is obtained (Table 2) but not to the same extent as in one precipitation by EPR alone. It should also be noted that while about 75% of the protein was lost from this EPR treated ethanol precipitate only about half of the radioactivity in the DNA was recovered. Precipitation of nucleic acids by ethanol is most commonly achieved by the addition of 2 or sometimes 2.5 volumes of the reagent. Higher concentrations may precipitate low molecular weight materials and even buffer salts. Ethanolic perchlorate reagent was formulated by trial and error such that on addition to an extract the final concentration of ethanol would be in the range 66-70% and the final perchlorate 25-30% (w/v) and also to keep at a minimum the volume it is necessary to add. It is suggested that since the efficiency of preparation of nucleic acid
68
J. WILCOCKSON
from tissues by this method is only limited by the efficiency of the extraction procedure itself and hardly at all by the EPR step that it may be useful in the chemical estimation of nucleic acids to free a crude extract of interfering substances prior to a colour test. ACKNOWLEDGMENTS This work was completed with the financial support of Deutsche Forschungsgemeinschaft (SFB 103). The advice, encouragement and provision of laboratory facilities by Professor Dietrich Werner of this University is also gratefully acknowledged.
REFERENCES 1. 2. 3. 4.
Wilcockson, J. (1973) Biochem. J. 135, 559-561. Wilcockson, J., and Hull, R. (1974) J. Gen. Viral. 23, 107-l 11. Miller, C. 0. (1967)Ann. N.Y. Acad. Sci. 144, 2.51-257. Marmur, J. (1961) J. Mol. Biol. 3, 208-218.
