ANALYTICAL BIOCHEMISTRY 70, 346-353 (1976)
Adsorbent Filters: A New Technique for Microexperimentation on Nucleic Acid MICHAEL YARUS
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80302 Received May 5, 1975; accepted September 11, 1975 When solutions of protein are filtered through nitrocellulose filters, the protein is retained on the membrane. Such protein-filters are a useful reagent; this paper shows that they adsorb a variety of nucleic acids, and that these may be re-eluted specifically. A variety of uses in collection, fractionation, and sensitive detection of nucleic acids is possible.
I have previously noted that almost all proteins stick to nitrocellulose membrane filters when a solution is passed through them (1). This paper indicates the potentially useful properties of filters so exposed to basic protein, which have become adsorbent for nucleic acids. METHODS
Filters are 24-mm Millipore (Bedford, Mass.) HA membrane filters for all experiments, save chromatography, where Millipore HA, 47-mm filters of 0.45 ~m pore size were used. Filters are made adsorbent by filtering 1-10 ml of an H20 solution of protein containing a total of 10 p.g to several milligrams (by weight). Application of nucleic acids to the filter as a whole is performed by filtrations of 1 ml of the corresponding H20 solution in a normal filter holder, usually the same holder in which the filter has just been assembled by prefiltration of protein. For chromatography, filters are prepared by a somewhat more complex procedure. A 47-mm filter is presoaked in 0.2 mg/ml cytochrome-c in H20, and then 10 ml of this solution is evenly filtered through. Cytochrome-c is the protein of choice for chromatography; it seems to give less irreversible binding at the origin than other proteins, and the cytochrome color conveniently reveals any unevenness in the protein deposit on the filter. Irreversible binding is further reduced by filtering 2 A260 of poly(A), removing the poly(A) with 10 ml of 0.50 M NaC104, and finally twice washing with 10 ml of 2% glycerol in H20. In these experiments, cytochrome c is from horse heart (type II); histone is from calf thymus (#H-9125); both are also from Sigma Chemical Co. (St. Louis, Missouri). Methylated albumin is a product of Worthington Biochemical Corp. 346 Copyright© 1976by AcademicPress, Inc. All rightsof reproductionin any formreserved.
ADSORBENT FILTERS
347
(Freehold, N.J.). Tritiated poly(U), poly(C), and poly(A) are from Miles Laboratories (Kankakee, Illinois), as are the unlabeled homopolynucleotides. [3H]Val-tRNA was made by aminoacylation of mixed tRNA from Escherichia coli (2) to a limit of 78 pmol/A260 with valine of specific activity 1750 cpm/pmol. [14C]E. coli RNA is total RNA derived from culture supplemented with [14C]uracil for several hours; as expected, and shown in other experiments, it consists predominantly of rRNA and tRNA. A sample to be chromatographed is applied by drawing a fine capillary pipet containing it across the filter between two origin marks about 1 cm apart. Suction is released, and the filter with its adsorbed line of sample at the origin is taken from its holder and cut into a rectangle whose long sides are at right angles to the origin line. It is immediately subjected to gradient thin-layer chromatography in the following way. The chromatography chamber is an overturned beaker whose atmosphere is humidified. Under the beaker is a piece of tygon tubing which carries the output from a linear gradient maker. The tubing is slit so that the origin end of the filter can be inserted in it, and, after the slit, several centimeters of tubing descend to a waste beaker. This latter (the descending tube) is important to prevent overflow of the elution fluid at the insertion point of the filter. A linear gradient from 0.01-0.40 M NaC104 in 2% glycerol is then pumped through the tubing so that the period of the gradient matches the time required for the front to transverse the filter. After chromatography, the filter was dried in air and cut into 1.5-mm sections; the sections are counted in a Beckman LS-100 scintillation counter. For sensitive detection of nucleic acid a special application technique was devised to make very small spots. Capillaries (Coming Cat. No. 7099-S, disposable micro-sampling pipets) were drawn out in a gas flame, and cut and lightly fire-polished so as to have a smooth, square tip of inside diameter of about 200 /xm. A known volume (usually 1 /zl) of an H20 solution ofisotopically labeled nucleic acid ([3H]poly(C) in the experiment in Fig. 4) was instilled into the capillary by tip-to-tip transfer from another volumetric capillary pipet (Drummond Scientific Co., Bromall, Pennsylvania). The nucleic acid solution also contained toluidine blue in order to mark the spot where the nucleic acid was absorbed. The fine-tipped capillaries, charged with the blue [3H]RNA solution, were held loosely and vertically in rows in a small jig made by drilling holes in a Lucite block. The jig is suspended above the filter, the latter freshly made and maintained under constant suction over a GF/C glass fiber filter of the same size (Reeve Angel, Clifton, N.J.). Under these conditions the liquid in the capillaries, which rest on the filter under their own weight, will transfer smoothly and completely to the filter, leaving a tiny spot. A light tap on the top of a balky capillary usually suffices to start transfer, if necessary. One hundred assays may be performed on a single filter, if desired. This assures that they will all be carried out under the same conditions. After application
348
MICHAEL YARUS
of the spots, the filter is removed, dried at room temperature, and mounted flat on a 25 × 75-mm microscope slide using double-sided Scotch tape (Minnesota Mining and Manufacturing, St. Paul, Minn.). The mounted filter is now dipped in liquid photographic emulsion (Kodak NTB-3), dried, and stored for a suitable time in a light tight box. It is developed for 3 min at 20° in Kodak D-19 developer, and fixed for 5 min in Kodak acid fixer. After washing, it is dried and prepared for examination by dipping in xylene and then applying a cover glass over Permount (Fisher Scientific Co., Fairlawn, N.J.). Grains are counted under oil immersion at 1000X, using a Whipple reticle to subdivide the optical field. A microscope with an intense light source is required to illuminate the opaque filter. RESULTS AND DISCUSSION Figure 1 exhibits the fraction of applied counts which are bound to several types of adsorbent filters at various levels of input RNA. In 1A, the filter is a hypothetical unit with a fixed total capacity for RNA. When more than capacity is applied, some passes through the filter, and the percentage retained sharply declines. The lower panels show that real filters behave similarly to the theoretical ideal, save that percentage cpm bound does not decline as sharply. It is as if there are sites of different affinity of RNA on the real filters, some of which are not occupied until the majority are past their capacity. This is particularly noticeable, for reasons which are unclear, when tRNA binds to histone filters (Fig. 1C). Different nucleic acids saturate the filters at different levels; this is probably due to differences in molecular size, at least in part. Differences in capacity suggest that absorbent filters could be used not only, e.g., to collect small amounts of nucleic acid from large volumes, but also perhaps to fractionate it. This is considered further below. All three types of filters have a capacity which is similar for tRNA, and histone and cytochrome-c filters are nearly identical in their capacity for poly (U). The number of sites created per microgram of protein filtered must be very much the same then, despite the other differences in the three proteins. The stack of points (at 1.4 A260 tRNA applied) in Fig. 1D represents the fraction bound when 10, 25,100,500, and 2000/~g of protein filter are applied, reading upward from the lowest point. Evidently, variation of the amount of methylated albumium over this range shifts the binding curve, and thus probably the capacity relatively little. In all experiments in Fig. 1, a control was included in which RNA was passed through a plain filter. These controls were always negative; the binding seen in Fig. 1 is therefore a property conferred on the filter by prefiltration of protein, and not a property of the filter material itself. It would sometimes be convenient to elute absorbed RNA, and data on elution with various solvents also gives an indication of the forces involved in binding. Thus, elution data is presented in Table I. Elution from histone
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FIG. 1. Retention of nucleic acid by an adsorbent filter. (A) Theoretical behavior of a uniform, perfectly adsorbent filter as input increases. (B) Fraction of poly(U) and tRNA bound to a cytochrome-c filter. (C) Fraction ofpoly(U), poly(C), poly(A), and tRNA bound to a histone filter. (D) Fraction of tRNA bound to a methylated-albumin filter.
and methylated albumin filters is similar. There may be a trend toward elution at lower pH, though pH's low enough to precipitate tRNA are ineffective (2 N H CI). Salt is quite effective, and the denaturing salt NaCIO4 (3) is much more effective than high concentrations of NaC1. These data suggest that a combination of ionic and hydrophobic forces bind tRNA to the filter. Sodium acetate and NaCIO4 apparently elute the RNA from the protein filter; they do not act by removing the protein itself because
350
MICHAEL YARUS TABLE 1 ELUTION OF TRNA FROM FILTERSa
tRNA remaining (%) Eluant
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H20 1 M K2HPO4-KH2PO4, p H 7.0 1 M T r i s - H C l , p H 8.9 1 M N a O A C , p H 5.5 2 M MgCI2 5 M NaCI 1 M NaC104 2 N HC1 1 N NaOH 95% Ethanol 2% SDS
100 22 99 -6 17 1 93 0 88 0
100 44 74 5 11 41 2 92 -85 0
a All filters were made by prefiltefing 100 g.g of protein, then 0.14 A2~0of tRNA. The filters were washed with 3 ml of the eluant at room temperature, then 3 ml of H20, and dried and counted in the scintillation counter. All percentages are normalized to filters washed with H20 only. A bar indicates that the measurement was not done. methylated albumin filters pretreated with 1 M NaOAc, p H 5.5, or 0.25 M NaCIO4 have the same capacity for tRNA as filters not treated with eluant (data not shown). Since these two salts seemed among the best and mildest eluants, progressive elution experiments using increasing concentrations of NaCIO4 and NaOAc, p H 5.5, were performed. The release of tRNA and of a mixture o f E . coli ribosomal and transfer RNA from methylated albumin filters is shown in Fig. 2. As expected from the data of Table I, perchlorate solutions give more quantitative elution of tRNA, but also yield a sharper transition at lower salt concentration (Fig. 2). Most important, when a mixture composed of total E. coli RNA's is applied, NaC104 elution produces a profile suggesting successive elution of several components with increasing affinities for the filter. The first of these components is released at the same NaCIO4 concentration as a single species of tRNA. This suggests even more strongly than Fig. 1 that absorbent filters can be used to fractionate, as well as to merely adsorb RNA. One could conceive of a stack of absorbent filters as a sort of modular column; a sample could be applied to a single filter, it could be placed on top of the stack, and after a suitable elution regime to move the sample into the stack, each filter could be r e m o v e d and treated as a fraction. H o w e v e r , the protocol actually adopted in order to demonstrate fractionation utilizes NaCIO4 solutions to move a sample, applied as a line at one side of the filter, across a single cytochrome-c filter by gradient
ADSORBENT
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thin-layer chromatography. The filter is then dried and sliced into 1.5-mm sections which are counted by scintillation. Figure 3 shows the result when a mixture of [3H]Val-tRNA and total E. coli [a4C]RNA was applied at the starting line and subjected to a gradient of 0 . 0 1 - 0 . 4 0 u NaCIO4. The mixture is indeed mobilized and fractionated as the front moves across the filter. There is 14C-stable RNA whose mobility is similar to the single-labeled [3H]amino acyl-tRNA, as expected. Twenty-eight percent of the total [14C]RNA moves as a leading peak; this is quite similar to the fraction of early eluting, tRNA-like material observed in Fig. 2. These data suggest that by choosing filtration conditions correctly, adsorbent filters could be used to select some species from a mixture of RNA's for separate examination, or to measure the amounts of several species originally applied as a single spot. As a final application, I should like to demonstrate that adsorbent filters
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352
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of nitrocellulose can be employed in an ultrasensitive radioassay for small amounts of nucleic acid. If radioactive nucleic acid is adsorbed to a tiny spot, and then the filter is coated with liquid photoemulsion, the number of grains counted microscopically above the spot, after exposure and development, should measure the radioactivity applied. A miniscule spot is required to minimize the number of background grains included, and therefore to maximize the sensitivity of the measurement. A method of making many spots a few hundred micrometers in diameter, each marked by a dye spot, on one 25-mm filter is described in Methods. Figure 4 is a plot of the density of grains counted above spots which contain varied amounts of radioactivity due to tritiated nucleic acid. Several things about this calibration curve are notable. First, the sensitivity, even with only one week exposure before development, is very good. Probably 10-~ cpm is detectable in this manner, and sensitivity (and standard deviation) in this experiment have not been optimized. A smaller spot could be used, the latent background present in the emulsion could be suppressed by exposure to an oxidizing vapor (4), development could be varied to maximize the signal to noise, and longer exposures are easily possible. Such tactics extend sensitivity to the order of 10-a cpm. Second, the overall response of the assay (Fig. 4) is much less than linear; in fact, observed grains increase approximately as applied radioactivity to the power 0.3. As a practical matter, this response gives the assay a very wide range, certainly 5 to 6 orders of magnitude. This is [O
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ADSORBENT FILTERS
353
probably an advantage in an assay which requires some time to perform, which uses a sensitive photographic element which has a limited response range, and in which the levels of radioactivity in individual spots will be varied and unknown in advance. This same characteristic, however, also limits the precision of individual measurements, and is disadvantageous in that respect. These data also raise the question: Why dependance on the 0.3 power? There seem to be two phenomena involved: both pivot on the fact that only electrons which reach the superficial layer of emulsion are counted. As more nucleic acid is applied by the capillary, it is adsorbed in a column which lengthens progressively below the spot of contact. Thus, as more sample is applied, the average 3H-atom is further and further from the surface, and its emitted electron is accordingly less and less likely to reach the surface and be counted instead of being absorbed by collision by filter atoms. The second factor is geometrical, and complements the first: as more and more material is applied, the average solid angle subtended by the fixed area of measurement (viewed from the standpoint of material deep in the filter) at the surface of the filter declines, and the fraction of emitted electrons passing through it does also. Thus, as more and more material is applied, it is counted progressively less and less efficiently, probably accounting for the characteristic response and range of the assay. This characteristic must be appreciated in any use of the technique to assay unknowns; more careful standardization is obviously required than when only a single, constant correction for self-absorption is required, as in most other 3H-counting techniques. Even more exotic combinations of techniques may be contemplated: chromatography and autoradiography could be combined, and a small spot of material could be fractionated by TLC or specific elution and then the distribution established by grain counting, so as to have a microfractionation method as well. An automatic grain-counting technique could be desirable for such an application because of the microscopically vast areas to be surveyed for grains.
ACKNOWLEDGMENT This work was supported by USPHS research Grant No. 15925.
REFERENCES 1. Yarus, M., and Berg, P. (1970)Anal. Biochem 35, 450. 2. Yarus, M. (1972) Biochemistry 11, 2352. 3. von Hippel, P. H., and Schleich, T. (1969)In Biological Macromolecules (Timosheff and Fasman, eds.), Vol. 2, p. 417, Marcel Dekker, New York. 4. Caro, L. G. (1964) In Methods in Cell Physiology (Prescott, D. M., ed.) Vol. 1, p. 327, Academic Press, New York.
Adsorbent Filters: A New Technique for Microexperimentation on Nucleic Acid MICHAEL YARUS
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80302 Received May 5, 1975; accepted September 11, 1975 When solutions of protein are filtered through nitrocellulose filters, the protein is retained on the membrane. Such protein-filters are a useful reagent; this paper shows that they adsorb a variety of nucleic acids, and that these may be re-eluted specifically. A variety of uses in collection, fractionation, and sensitive detection of nucleic acids is possible.
I have previously noted that almost all proteins stick to nitrocellulose membrane filters when a solution is passed through them (1). This paper indicates the potentially useful properties of filters so exposed to basic protein, which have become adsorbent for nucleic acids. METHODS
Filters are 24-mm Millipore (Bedford, Mass.) HA membrane filters for all experiments, save chromatography, where Millipore HA, 47-mm filters of 0.45 ~m pore size were used. Filters are made adsorbent by filtering 1-10 ml of an H20 solution of protein containing a total of 10 p.g to several milligrams (by weight). Application of nucleic acids to the filter as a whole is performed by filtrations of 1 ml of the corresponding H20 solution in a normal filter holder, usually the same holder in which the filter has just been assembled by prefiltration of protein. For chromatography, filters are prepared by a somewhat more complex procedure. A 47-mm filter is presoaked in 0.2 mg/ml cytochrome-c in H20, and then 10 ml of this solution is evenly filtered through. Cytochrome-c is the protein of choice for chromatography; it seems to give less irreversible binding at the origin than other proteins, and the cytochrome color conveniently reveals any unevenness in the protein deposit on the filter. Irreversible binding is further reduced by filtering 2 A260 of poly(A), removing the poly(A) with 10 ml of 0.50 M NaC104, and finally twice washing with 10 ml of 2% glycerol in H20. In these experiments, cytochrome c is from horse heart (type II); histone is from calf thymus (#H-9125); both are also from Sigma Chemical Co. (St. Louis, Missouri). Methylated albumin is a product of Worthington Biochemical Corp. 346 Copyright© 1976by AcademicPress, Inc. All rightsof reproductionin any formreserved.
ADSORBENT FILTERS
347
(Freehold, N.J.). Tritiated poly(U), poly(C), and poly(A) are from Miles Laboratories (Kankakee, Illinois), as are the unlabeled homopolynucleotides. [3H]Val-tRNA was made by aminoacylation of mixed tRNA from Escherichia coli (2) to a limit of 78 pmol/A260 with valine of specific activity 1750 cpm/pmol. [14C]E. coli RNA is total RNA derived from culture supplemented with [14C]uracil for several hours; as expected, and shown in other experiments, it consists predominantly of rRNA and tRNA. A sample to be chromatographed is applied by drawing a fine capillary pipet containing it across the filter between two origin marks about 1 cm apart. Suction is released, and the filter with its adsorbed line of sample at the origin is taken from its holder and cut into a rectangle whose long sides are at right angles to the origin line. It is immediately subjected to gradient thin-layer chromatography in the following way. The chromatography chamber is an overturned beaker whose atmosphere is humidified. Under the beaker is a piece of tygon tubing which carries the output from a linear gradient maker. The tubing is slit so that the origin end of the filter can be inserted in it, and, after the slit, several centimeters of tubing descend to a waste beaker. This latter (the descending tube) is important to prevent overflow of the elution fluid at the insertion point of the filter. A linear gradient from 0.01-0.40 M NaC104 in 2% glycerol is then pumped through the tubing so that the period of the gradient matches the time required for the front to transverse the filter. After chromatography, the filter was dried in air and cut into 1.5-mm sections; the sections are counted in a Beckman LS-100 scintillation counter. For sensitive detection of nucleic acid a special application technique was devised to make very small spots. Capillaries (Coming Cat. No. 7099-S, disposable micro-sampling pipets) were drawn out in a gas flame, and cut and lightly fire-polished so as to have a smooth, square tip of inside diameter of about 200 /xm. A known volume (usually 1 /zl) of an H20 solution ofisotopically labeled nucleic acid ([3H]poly(C) in the experiment in Fig. 4) was instilled into the capillary by tip-to-tip transfer from another volumetric capillary pipet (Drummond Scientific Co., Bromall, Pennsylvania). The nucleic acid solution also contained toluidine blue in order to mark the spot where the nucleic acid was absorbed. The fine-tipped capillaries, charged with the blue [3H]RNA solution, were held loosely and vertically in rows in a small jig made by drilling holes in a Lucite block. The jig is suspended above the filter, the latter freshly made and maintained under constant suction over a GF/C glass fiber filter of the same size (Reeve Angel, Clifton, N.J.). Under these conditions the liquid in the capillaries, which rest on the filter under their own weight, will transfer smoothly and completely to the filter, leaving a tiny spot. A light tap on the top of a balky capillary usually suffices to start transfer, if necessary. One hundred assays may be performed on a single filter, if desired. This assures that they will all be carried out under the same conditions. After application
348
MICHAEL YARUS
of the spots, the filter is removed, dried at room temperature, and mounted flat on a 25 × 75-mm microscope slide using double-sided Scotch tape (Minnesota Mining and Manufacturing, St. Paul, Minn.). The mounted filter is now dipped in liquid photographic emulsion (Kodak NTB-3), dried, and stored for a suitable time in a light tight box. It is developed for 3 min at 20° in Kodak D-19 developer, and fixed for 5 min in Kodak acid fixer. After washing, it is dried and prepared for examination by dipping in xylene and then applying a cover glass over Permount (Fisher Scientific Co., Fairlawn, N.J.). Grains are counted under oil immersion at 1000X, using a Whipple reticle to subdivide the optical field. A microscope with an intense light source is required to illuminate the opaque filter. RESULTS AND DISCUSSION Figure 1 exhibits the fraction of applied counts which are bound to several types of adsorbent filters at various levels of input RNA. In 1A, the filter is a hypothetical unit with a fixed total capacity for RNA. When more than capacity is applied, some passes through the filter, and the percentage retained sharply declines. The lower panels show that real filters behave similarly to the theoretical ideal, save that percentage cpm bound does not decline as sharply. It is as if there are sites of different affinity of RNA on the real filters, some of which are not occupied until the majority are past their capacity. This is particularly noticeable, for reasons which are unclear, when tRNA binds to histone filters (Fig. 1C). Different nucleic acids saturate the filters at different levels; this is probably due to differences in molecular size, at least in part. Differences in capacity suggest that absorbent filters could be used not only, e.g., to collect small amounts of nucleic acid from large volumes, but also perhaps to fractionate it. This is considered further below. All three types of filters have a capacity which is similar for tRNA, and histone and cytochrome-c filters are nearly identical in their capacity for poly (U). The number of sites created per microgram of protein filtered must be very much the same then, despite the other differences in the three proteins. The stack of points (at 1.4 A260 tRNA applied) in Fig. 1D represents the fraction bound when 10, 25,100,500, and 2000/~g of protein filter are applied, reading upward from the lowest point. Evidently, variation of the amount of methylated albumium over this range shifts the binding curve, and thus probably the capacity relatively little. In all experiments in Fig. 1, a control was included in which RNA was passed through a plain filter. These controls were always negative; the binding seen in Fig. 1 is therefore a property conferred on the filter by prefiltration of protein, and not a property of the filter material itself. It would sometimes be convenient to elute absorbed RNA, and data on elution with various solvents also gives an indication of the forces involved in binding. Thus, elution data is presented in Table I. Elution from histone
349
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and methylated albumin filters is similar. There may be a trend toward elution at lower pH, though pH's low enough to precipitate tRNA are ineffective (2 N H CI). Salt is quite effective, and the denaturing salt NaCIO4 (3) is much more effective than high concentrations of NaC1. These data suggest that a combination of ionic and hydrophobic forces bind tRNA to the filter. Sodium acetate and NaCIO4 apparently elute the RNA from the protein filter; they do not act by removing the protein itself because
350
MICHAEL YARUS TABLE 1 ELUTION OF TRNA FROM FILTERSa
tRNA remaining (%) Eluant
Histone Filter
Me-BSAFilter
H20 1 M K2HPO4-KH2PO4, p H 7.0 1 M T r i s - H C l , p H 8.9 1 M N a O A C , p H 5.5 2 M MgCI2 5 M NaCI 1 M NaC104 2 N HC1 1 N NaOH 95% Ethanol 2% SDS
100 22 99 -6 17 1 93 0 88 0
100 44 74 5 11 41 2 92 -85 0
a All filters were made by prefiltefing 100 g.g of protein, then 0.14 A2~0of tRNA. The filters were washed with 3 ml of the eluant at room temperature, then 3 ml of H20, and dried and counted in the scintillation counter. All percentages are normalized to filters washed with H20 only. A bar indicates that the measurement was not done. methylated albumin filters pretreated with 1 M NaOAc, p H 5.5, or 0.25 M NaCIO4 have the same capacity for tRNA as filters not treated with eluant (data not shown). Since these two salts seemed among the best and mildest eluants, progressive elution experiments using increasing concentrations of NaCIO4 and NaOAc, p H 5.5, were performed. The release of tRNA and of a mixture o f E . coli ribosomal and transfer RNA from methylated albumin filters is shown in Fig. 2. As expected from the data of Table I, perchlorate solutions give more quantitative elution of tRNA, but also yield a sharper transition at lower salt concentration (Fig. 2). Most important, when a mixture composed of total E. coli RNA's is applied, NaC104 elution produces a profile suggesting successive elution of several components with increasing affinities for the filter. The first of these components is released at the same NaCIO4 concentration as a single species of tRNA. This suggests even more strongly than Fig. 1 that absorbent filters can be used to fractionate, as well as to merely adsorb RNA. One could conceive of a stack of absorbent filters as a sort of modular column; a sample could be applied to a single filter, it could be placed on top of the stack, and after a suitable elution regime to move the sample into the stack, each filter could be r e m o v e d and treated as a fraction. H o w e v e r , the protocol actually adopted in order to demonstrate fractionation utilizes NaCIO4 solutions to move a sample, applied as a line at one side of the filter, across a single cytochrome-c filter by gradient
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thin-layer chromatography. The filter is then dried and sliced into 1.5-mm sections which are counted by scintillation. Figure 3 shows the result when a mixture of [3H]Val-tRNA and total E. coli [a4C]RNA was applied at the starting line and subjected to a gradient of 0 . 0 1 - 0 . 4 0 u NaCIO4. The mixture is indeed mobilized and fractionated as the front moves across the filter. There is 14C-stable RNA whose mobility is similar to the single-labeled [3H]amino acyl-tRNA, as expected. Twenty-eight percent of the total [14C]RNA moves as a leading peak; this is quite similar to the fraction of early eluting, tRNA-like material observed in Fig. 2. These data suggest that by choosing filtration conditions correctly, adsorbent filters could be used to select some species from a mixture of RNA's for separate examination, or to measure the amounts of several species originally applied as a single spot. As a final application, I should like to demonstrate that adsorbent filters
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352
MICHAEL YARUS
of nitrocellulose can be employed in an ultrasensitive radioassay for small amounts of nucleic acid. If radioactive nucleic acid is adsorbed to a tiny spot, and then the filter is coated with liquid photoemulsion, the number of grains counted microscopically above the spot, after exposure and development, should measure the radioactivity applied. A miniscule spot is required to minimize the number of background grains included, and therefore to maximize the sensitivity of the measurement. A method of making many spots a few hundred micrometers in diameter, each marked by a dye spot, on one 25-mm filter is described in Methods. Figure 4 is a plot of the density of grains counted above spots which contain varied amounts of radioactivity due to tritiated nucleic acid. Several things about this calibration curve are notable. First, the sensitivity, even with only one week exposure before development, is very good. Probably 10-~ cpm is detectable in this manner, and sensitivity (and standard deviation) in this experiment have not been optimized. A smaller spot could be used, the latent background present in the emulsion could be suppressed by exposure to an oxidizing vapor (4), development could be varied to maximize the signal to noise, and longer exposures are easily possible. Such tactics extend sensitivity to the order of 10-a cpm. Second, the overall response of the assay (Fig. 4) is much less than linear; in fact, observed grains increase approximately as applied radioactivity to the power 0.3. As a practical matter, this response gives the assay a very wide range, certainly 5 to 6 orders of magnitude. This is [O
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FIG. 4. Photographic grain density above spots containing various amounts of poly(C).
Grains were counted after 1-weekexposure; the area unit was definedby a Whipple reticle in the ocular of the microscope used to count grains; background was about 0.52 grains per area. CPM applied were determined by acid precipitation of an aliquot and counting in a scintillation counter at 25% efficiency.
ADSORBENT FILTERS
353
probably an advantage in an assay which requires some time to perform, which uses a sensitive photographic element which has a limited response range, and in which the levels of radioactivity in individual spots will be varied and unknown in advance. This same characteristic, however, also limits the precision of individual measurements, and is disadvantageous in that respect. These data also raise the question: Why dependance on the 0.3 power? There seem to be two phenomena involved: both pivot on the fact that only electrons which reach the superficial layer of emulsion are counted. As more nucleic acid is applied by the capillary, it is adsorbed in a column which lengthens progressively below the spot of contact. Thus, as more sample is applied, the average 3H-atom is further and further from the surface, and its emitted electron is accordingly less and less likely to reach the surface and be counted instead of being absorbed by collision by filter atoms. The second factor is geometrical, and complements the first: as more and more material is applied, the average solid angle subtended by the fixed area of measurement (viewed from the standpoint of material deep in the filter) at the surface of the filter declines, and the fraction of emitted electrons passing through it does also. Thus, as more and more material is applied, it is counted progressively less and less efficiently, probably accounting for the characteristic response and range of the assay. This characteristic must be appreciated in any use of the technique to assay unknowns; more careful standardization is obviously required than when only a single, constant correction for self-absorption is required, as in most other 3H-counting techniques. Even more exotic combinations of techniques may be contemplated: chromatography and autoradiography could be combined, and a small spot of material could be fractionated by TLC or specific elution and then the distribution established by grain counting, so as to have a microfractionation method as well. An automatic grain-counting technique could be desirable for such an application because of the microscopically vast areas to be surveyed for grains.
ACKNOWLEDGMENT This work was supported by USPHS research Grant No. 15925.
REFERENCES 1. Yarus, M., and Berg, P. (1970)Anal. Biochem 35, 450. 2. Yarus, M. (1972) Biochemistry 11, 2352. 3. von Hippel, P. H., and Schleich, T. (1969)In Biological Macromolecules (Timosheff and Fasman, eds.), Vol. 2, p. 417, Marcel Dekker, New York. 4. Caro, L. G. (1964) In Methods in Cell Physiology (Prescott, D. M., ed.) Vol. 1, p. 327, Academic Press, New York.
