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Dialysis of small samples in agarose gels
ANALYTICAL
BIOCHEMISTRY
123, 83-85
(1982)
Dialysis of Small Samples in Agarose DAVID Department
of Biochemistry,
FREIFELDER Brandeis
AND MARC University,
Gels
BETTER
Waltham,
Massachusetts
02254
Received February 4. 1982 Reduction of the salt concentration of small volumes (0.1 ml or less) of solutions of macromolecules can be accomplished by placing the solution in a small well in an agarose gel. This technique has been especially useful in preparation of samples for electron microscopy.
The need to reduce the ionic strength or to change the buffer composition of a solution of macromolecules often arises. Various simple techniques, such as dialysis through a semipermeable membrane or desalting with columns of dextran beads, are available, but if the sample size is 0.1 ml or less, these standard techniques prove difficult and result in loss of a significant fraction of the sample. In our laboratory we have repeatedly had to remove CsCl and other salts from very small samples in preparation for electron microscopy of DNA and thus have developed a rapid procedure that entails very little sample loss. This procedure is described in this report.
necessary to remove a small amount of water that accumulates in the well. The change in salt concentration as a function of time was measured in two ways. CsCl concentrations were measured by removing a 204 sample and weighing it to obtain the solution density. The concentration was then derived from standard concentration-density tables. Concentrations of other solutions were determined by measuring the electrical conductivity of the sample and comparing it to standard curves of conductivity vs concentration obtained by measuring solutions of known concentrations. RESULTS
MATERIALS AND METHODS
Large macromolecules such as DNA penetrate 1% agarose gels very slowly, if at all, unless an electric field is applied. Inorganic ions, however, enter the gel freely and rapidly so that ions can be removed from a nucleic acid solution by placing a sample in a small well in an agarose gel and allowing the salts to diffuse into the gel. The rate of decrease of the salt concentration will depend on a variety of factors, such as the gel concentration, the size of the ions, and the surface-to-volume ratio of the well. Figure 1 shows the decrease of CsCl concentration with time for a 0.2-ml sample placed in a well in 100 ml of 1% agarose. The concentration dropped from 6.5 to 2 M in 20 min and to 0.85 M in 60 min. When
Agarose was purchased from Sea-Kern and prepared as a 1% solution in water of 10 mM Tris, pH 8, 5 mM EDTA, and allowed to harden. When ready to use, the gel was melted and poured into a test tube or a small beaker and allowed to harden again. Stock gels have been repeatedly melted and hardened up to 20 times. Wells in the agarose are prepared by filling a 15 X IOO-mm test tube or a loo-ml beaker with liquid 1% agarose solution in which a glass rod 3-5 mm in diameter is suspended. Once the agarose has hardened, the rod is removed leaving a well. For O.lml samples the well is made 10 to 15 mm deep. Before adding the sample it is usually 83
0003-2697/82/090083-03$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
84
FREIFELDER
0
I
I
1
1
I
I
IO
20
30
40
50
60
Time
(M~nuies)
FIG. 1. Decrease in molarity of CsCl when 0.2 ml of 6.5 M CsCl is placed in a well in 100 ml of 1% agarose (0). The closed triangle represents the final concentration of CsCl after two successive dialyses for 10 min.
a sample was transferred after 10 min to a second well in fresh agarose, 0.85 M was achieved in 20 min total time. After 15 h in the first well the concentration reached 0.01 M; the equilibrium value for the relative volumes in this experiment would be 0.006 M.
AND BETTER
To test for possible loss of DNA during desalting, radioactive Escherichia coli phage X DNA was added; there was no detectable loss of recoverable DNA after 20 min and no more than 5% after 16 h. For our purpose, which was to lower the concentration of CsCl so that DNA samples from a CsCl density gradient could be spread for electron microscopy by the Kleinschmidt procedure, the protocol with the two IO-min incubations has been used. Figure 2a shows data for several other salts at different initial concentrations. Surprisingly, the concentrations seem to drop approximately in a linear manner with time during the first 30 min. Also, the concentration decrease is roughly twofold whether the initial concentration is 0.01, 0.1, or 1 M. Since it is often desirable to drop the concentration several loo-fold, we tested on the 1 M NaCl sample the effect of transfers of the sample every 60 min to a fresh well in an unused block of agarose. As shown in Fig. 2b there is a multiplicative effect, as would be expected. This protocol accelerates the salt-removal process, exactly as in standard dialysis. After three transfers to loo-ml volumes a final overnight dialysis reduced the concentration more than 104-fold. b
o.ool
1
~;“”
or;OC12
,
,o-4 1 IO 20 Time in Minutes
30
, , , ;; p4 I 2 3 Time I” Hours
16
FIG. 2. Dialysis of various salt solutions in agarose. (a) Three different starting concentrations. The NaCl and CaC& curves are usually indistinguishable. (b) At t = 1, 2, and 3 h a NaCl solution was transferred to a well in a fresh block of agarose.
DIALYSIS
OF SMALL
SAMPLES
We have not tested other concentrations of agarose nor the degree of loss with small proteins. However, less than 10% of the activity of P-galactosidase, a large protein, is lost after 10 h and three transfers of 0.2-ml samples. This technique has also been used to remove sucrose from fractions obtained from sucrose density gradients. We have not measured the rate of decrease of the concentration of sucrose but have observed that a 20-min treatment is sufficient to enable any fraction to be layered again on a 5 to 20% sucrose density gradient. DISCUSSION
We have described a simple technique for removal of salts from small volume of solutions of macromolecules. There is little or no sample loss for O.l-ml samples of DNA nor presumably for large proteins either. This procedure should be useful in preparing samples for electron microscopy, gel electrophoresis, and centrifugation on sucrose gradients. We have used it routinely in desalting samples obtained from CsCl gradients. It is important to realize that there is a practical difference between dialysis through cellophane membranes and the agarose technique described in this paper. Ordinary dialysis tu ing is impermeable to molecules whose mo$ cular weight is above a few thousand. The high-permeability tubing, such as Spectropor, has a molecular weight cut off of about 20,000. However, in gel electrophoresis a 1% agarose gel is certainly permeable to molecules with molecular weights in the millions, although in the absence of the driving force of an electric field, penetration is exceedingly slow. Therefore, if one is using the agarose-dialysis technique with molecules of small excluded volume, such as small viruses or low-molecular-weight proteins, it is necessary to ascertain that the molecule of interest is not lost in the gel. If it is, the agarose technique can be modified by increasing the concentration of agarose. In recent years several other methods have been reported for dialysis of small volumes.
IN AGAROSE
GELS
85
Each of these methods has advantages and disadvantages. Two of them ( 1,2) use microchambers and standard dialysis tubing. These two methods have the advantages that dialysis is carried out in a standard way and losses are small, but the disadvantages that the chambers have to be fabricated and cleaned and may be cumbersome to assemble. If, however, dialysis is done very frequently, the cost of fabrication may be less than the cost of agarose, which is not easily reusable. Two other methods utilize a tiny piece of dialysis tubing either floating on a liquid surface (3) or placed on glass wool through which liquid flows by a combination of capillarity and siphoning (4). A droplet of the solution to be dialyzed is placed on the tubing; hence, the technique is called drop dialysis. These two procedures are also useful, although minimizing evaporation of tiny samples can be a problem. A fifth method (5) employs modified microtiter plates and is excellent when a large number of samples must be dialyzed. In many cases, the choice of method may be a matter of taste. There is, however, an important advantage of the agarose-dialysis procedure described in this paper-namely, that losses due to adsorption will rarely occur; such losses are common when solutions of macromolecules at very low concentrations are dialyzed with nitrocellulose tubing. ACKNOWLEDGMENTS This work was supported by Grant GM 14358 awarded to D.F. We wish to thank Chris Miller and Michael White for the instruction and use of a conductivity meter and Les Lang for performing the conductivity measurements This is publication No. 1401 of the Department of Biochemistry, Brandeis University.
REFERENCES 1. Brand, P. H., and Stansbury, R. (1979) Anal. Biothem. 94, 109-l 16. 2. Armstrong, I. L., and Tate, W. P. (1980) Anal. Biochem. 106, 469-410. 3. Marusyk, R., and Sergeant, A. (1980) Anal. Biothem. 105,403-404. 4. Awdeh, Z. L. (1976) Anal. Biochem. 71,601-602. 5. Budowle, B., Acton, R. T., and Barger, B. 0. ( 198 I ) Anal. Biochem. 118, 399-400.
BIOCHEMISTRY
123, 83-85
(1982)
Dialysis of Small Samples in Agarose DAVID Department
of Biochemistry,
FREIFELDER Brandeis
AND MARC University,
Gels
BETTER
Waltham,
Massachusetts
02254
Received February 4. 1982 Reduction of the salt concentration of small volumes (0.1 ml or less) of solutions of macromolecules can be accomplished by placing the solution in a small well in an agarose gel. This technique has been especially useful in preparation of samples for electron microscopy.
The need to reduce the ionic strength or to change the buffer composition of a solution of macromolecules often arises. Various simple techniques, such as dialysis through a semipermeable membrane or desalting with columns of dextran beads, are available, but if the sample size is 0.1 ml or less, these standard techniques prove difficult and result in loss of a significant fraction of the sample. In our laboratory we have repeatedly had to remove CsCl and other salts from very small samples in preparation for electron microscopy of DNA and thus have developed a rapid procedure that entails very little sample loss. This procedure is described in this report.
necessary to remove a small amount of water that accumulates in the well. The change in salt concentration as a function of time was measured in two ways. CsCl concentrations were measured by removing a 204 sample and weighing it to obtain the solution density. The concentration was then derived from standard concentration-density tables. Concentrations of other solutions were determined by measuring the electrical conductivity of the sample and comparing it to standard curves of conductivity vs concentration obtained by measuring solutions of known concentrations. RESULTS
MATERIALS AND METHODS
Large macromolecules such as DNA penetrate 1% agarose gels very slowly, if at all, unless an electric field is applied. Inorganic ions, however, enter the gel freely and rapidly so that ions can be removed from a nucleic acid solution by placing a sample in a small well in an agarose gel and allowing the salts to diffuse into the gel. The rate of decrease of the salt concentration will depend on a variety of factors, such as the gel concentration, the size of the ions, and the surface-to-volume ratio of the well. Figure 1 shows the decrease of CsCl concentration with time for a 0.2-ml sample placed in a well in 100 ml of 1% agarose. The concentration dropped from 6.5 to 2 M in 20 min and to 0.85 M in 60 min. When
Agarose was purchased from Sea-Kern and prepared as a 1% solution in water of 10 mM Tris, pH 8, 5 mM EDTA, and allowed to harden. When ready to use, the gel was melted and poured into a test tube or a small beaker and allowed to harden again. Stock gels have been repeatedly melted and hardened up to 20 times. Wells in the agarose are prepared by filling a 15 X IOO-mm test tube or a loo-ml beaker with liquid 1% agarose solution in which a glass rod 3-5 mm in diameter is suspended. Once the agarose has hardened, the rod is removed leaving a well. For O.lml samples the well is made 10 to 15 mm deep. Before adding the sample it is usually 83
0003-2697/82/090083-03$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
84
FREIFELDER
0
I
I
1
1
I
I
IO
20
30
40
50
60
Time
(M~nuies)
FIG. 1. Decrease in molarity of CsCl when 0.2 ml of 6.5 M CsCl is placed in a well in 100 ml of 1% agarose (0). The closed triangle represents the final concentration of CsCl after two successive dialyses for 10 min.
a sample was transferred after 10 min to a second well in fresh agarose, 0.85 M was achieved in 20 min total time. After 15 h in the first well the concentration reached 0.01 M; the equilibrium value for the relative volumes in this experiment would be 0.006 M.
AND BETTER
To test for possible loss of DNA during desalting, radioactive Escherichia coli phage X DNA was added; there was no detectable loss of recoverable DNA after 20 min and no more than 5% after 16 h. For our purpose, which was to lower the concentration of CsCl so that DNA samples from a CsCl density gradient could be spread for electron microscopy by the Kleinschmidt procedure, the protocol with the two IO-min incubations has been used. Figure 2a shows data for several other salts at different initial concentrations. Surprisingly, the concentrations seem to drop approximately in a linear manner with time during the first 30 min. Also, the concentration decrease is roughly twofold whether the initial concentration is 0.01, 0.1, or 1 M. Since it is often desirable to drop the concentration several loo-fold, we tested on the 1 M NaCl sample the effect of transfers of the sample every 60 min to a fresh well in an unused block of agarose. As shown in Fig. 2b there is a multiplicative effect, as would be expected. This protocol accelerates the salt-removal process, exactly as in standard dialysis. After three transfers to loo-ml volumes a final overnight dialysis reduced the concentration more than 104-fold. b
o.ool
1
~;“”
or;OC12
,
,o-4 1 IO 20 Time in Minutes
30
, , , ;; p4 I 2 3 Time I” Hours
16
FIG. 2. Dialysis of various salt solutions in agarose. (a) Three different starting concentrations. The NaCl and CaC& curves are usually indistinguishable. (b) At t = 1, 2, and 3 h a NaCl solution was transferred to a well in a fresh block of agarose.
DIALYSIS
OF SMALL
SAMPLES
We have not tested other concentrations of agarose nor the degree of loss with small proteins. However, less than 10% of the activity of P-galactosidase, a large protein, is lost after 10 h and three transfers of 0.2-ml samples. This technique has also been used to remove sucrose from fractions obtained from sucrose density gradients. We have not measured the rate of decrease of the concentration of sucrose but have observed that a 20-min treatment is sufficient to enable any fraction to be layered again on a 5 to 20% sucrose density gradient. DISCUSSION
We have described a simple technique for removal of salts from small volume of solutions of macromolecules. There is little or no sample loss for O.l-ml samples of DNA nor presumably for large proteins either. This procedure should be useful in preparing samples for electron microscopy, gel electrophoresis, and centrifugation on sucrose gradients. We have used it routinely in desalting samples obtained from CsCl gradients. It is important to realize that there is a practical difference between dialysis through cellophane membranes and the agarose technique described in this paper. Ordinary dialysis tu ing is impermeable to molecules whose mo$ cular weight is above a few thousand. The high-permeability tubing, such as Spectropor, has a molecular weight cut off of about 20,000. However, in gel electrophoresis a 1% agarose gel is certainly permeable to molecules with molecular weights in the millions, although in the absence of the driving force of an electric field, penetration is exceedingly slow. Therefore, if one is using the agarose-dialysis technique with molecules of small excluded volume, such as small viruses or low-molecular-weight proteins, it is necessary to ascertain that the molecule of interest is not lost in the gel. If it is, the agarose technique can be modified by increasing the concentration of agarose. In recent years several other methods have been reported for dialysis of small volumes.
IN AGAROSE
GELS
85
Each of these methods has advantages and disadvantages. Two of them ( 1,2) use microchambers and standard dialysis tubing. These two methods have the advantages that dialysis is carried out in a standard way and losses are small, but the disadvantages that the chambers have to be fabricated and cleaned and may be cumbersome to assemble. If, however, dialysis is done very frequently, the cost of fabrication may be less than the cost of agarose, which is not easily reusable. Two other methods utilize a tiny piece of dialysis tubing either floating on a liquid surface (3) or placed on glass wool through which liquid flows by a combination of capillarity and siphoning (4). A droplet of the solution to be dialyzed is placed on the tubing; hence, the technique is called drop dialysis. These two procedures are also useful, although minimizing evaporation of tiny samples can be a problem. A fifth method (5) employs modified microtiter plates and is excellent when a large number of samples must be dialyzed. In many cases, the choice of method may be a matter of taste. There is, however, an important advantage of the agarose-dialysis procedure described in this paper-namely, that losses due to adsorption will rarely occur; such losses are common when solutions of macromolecules at very low concentrations are dialyzed with nitrocellulose tubing. ACKNOWLEDGMENTS This work was supported by Grant GM 14358 awarded to D.F. We wish to thank Chris Miller and Michael White for the instruction and use of a conductivity meter and Les Lang for performing the conductivity measurements This is publication No. 1401 of the Department of Biochemistry, Brandeis University.
REFERENCES 1. Brand, P. H., and Stansbury, R. (1979) Anal. Biothem. 94, 109-l 16. 2. Armstrong, I. L., and Tate, W. P. (1980) Anal. Biochem. 106, 469-410. 3. Marusyk, R., and Sergeant, A. (1980) Anal. Biothem. 105,403-404. 4. Awdeh, Z. L. (1976) Anal. Biochem. 71,601-602. 5. Budowle, B., Acton, R. T., and Barger, B. 0. ( 198 I ) Anal. Biochem. 118, 399-400.