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A simple device for transverse slicing of polyacrylamide gel rods
ANALYTICAL
56, 571-575 (1973)
BIOCHEMISTRY
SHORT
A Simple
Device
COMMUNICATIONAL
for
Transverse
of Polyacrylamide
Gel
Slicing
Rods
The use of electrophoresis on polyacrylamide gel has permitted remarkable resolutions of macromolecular mixtures (i-3). A number of successful methods to fractionate gel rods or gel slabs for enzymic assay and for radioactivity assay have been described. A slicer constructed with a series of wires, which resembles a cheese cutter, is suitable for slicing gels of medium acrylamide concentration, i.e., 5-15% (4). A bolted series of razor blades is adequate to slice slabs (5) and is also useful in slicing cylindrical gel rods (6) and square gel columns (7). Fractionation of a gel rod in a sequential fashion has been attained either with microtome-like slicers (8-11) or with gel extrusion system through a precision syringe and a fixed needle into a stream of eluting liquid (12). With improved microtomes Iandolo (10) and Sheu and Ries (11) have made it possible to section soft gels without freezing. Several difficulties, however, have arisen with these fractionation methods. With devices using wires or blades, distortion and breakage of gels often take place by the pressure on gels. Although this difficulty is partly reduced with the circular blade model of Goldberger (13)) it is required to freeze gels, especially of low acrylamide concentration, rock hard prior to slicing with these devices. In practice, however, the distortion of gels during freezing and the adherence of gel slices to cold met,al blades interfere. An automatic fractionation is possible with the gel extrusion system; however, this system may not be universally applicable because of the difficulty with hard gels. A difficulty with the sequential fractionation is that the fitness of a gel rod to the holder influences t’he precision of slicing (10, 11). In the present paper a method of gel slicing by the use of a razor blade assembly is described. This method overcomes some of these disadvantages mentioned above, because no freezing prior to slicing is required, gel slices are obtainable in a separate state from each other, and slices are obtainable from soft gels as well as from hard gels. 571 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
572
SHORT
COMMUNICATIONS
1. Photographs of gel slicers in assembly and in use: (a) A pair of bolts on a wooden block and razor blades mounted to form a razor blade assembly. Mounting spacer rings between the blades of the assembly. (c) A sample gel inserted in the holes of spacer rings. (d) Pushing the assembly down on the gel (e) Taking a ring out with a gel slice inside.
FIG.
put (h) rod rod.
A razor blade assembly was made as follows: Double-edge blades of stainless steel (Hi-Stainless, Feather Safety Razor Co., Osaka, Japan) were rinsed with ethyl acetate to remove wax, then with water, and dried. About 60 pieces of the blades, being separated from each other were connected with a pair of screw by 1 mm thick plastic washers, bolts and nuts. Use of a wooden block with two holes to support the
SHORT
COMMUNICATIONS
573
bolts in the vertical and parallel position helped this procedure (Fig. la). Fift,y plastic spacer rings, each of which was 1 mm thick and had a hole 6 mm in diameter, were put by the use of pincers into the gaps of the blades (Fig. lb). The spacer rings should be placed on the blade assembly so that. the hole of each ring might be free from t,he blade edge. The position of each ring on the blade assembly was adjusted by insertion of a plastic rod through the spacer rings so that a gel rod might be inserted smoothly through the holes. The assembly was again put on the wooden block, and a sample gel rod, 50 mm long and 5 mm in diameter, was inserted vertically into the core of the stack of spacer rings. The assembly should be wetted with water prior to inserting soft gels. No special lubricant was necessary for inserting rods. The fitness of a rod to the hole of spacer rings was loose. After a gel rod was inserted in the right position (Fi g. lc) , the tightness of connection of the blades should be controlled by driving the nuts in order that the spacer rings smoothly moved into the gaps. The rings with the gel rod inside the holes were pushed at once into the gaps of blades, until the holes disappeared into the gaps, while a part of each ring still appeared in the blades. This procedure was attained by turning the assembly upside down on the bench and pushing it by both ends of bolts (Fig. Id). Although the handling is not dangerous, it is preferable to cover the other side of blade edges by something like an aluminum plate, or a plastic sheet. After the pushing, each spacer ring with a sliced gel inside the hole was pulled out, by a pair of pincers (Fig. le), and the gel slice was transferred into a test tube. We separated a slice from a ring by the use of a needle or by immersing the ring and gel slice in a
574
SHORT
COMMUNICATIONS
small amount of buffer solution. The blade assembly was rinsed with water and dried for the next use. It took 5-10 min to slice a gel rod and to transfer slices into test tubes. For the determination of slicing precision, gel rods of 3.75% w/v acrylamide (0.1% w/v N,N’-methylenebisacrylamide), 7.5% (0.2%), 15% (0.4%) and 30% (0.8%) concentrations, respectively, were sliced. Every gel rod was sliced through the entire length. The slice weight, however, may not quantitatively represent the slice thickness for the few slices from both ends of a gel rod, because the terminal slice may be less thick t.han the distance between adjacent two blades, and because a gel rod often swells at the ends so that one or two slices from the end region may weigh more than the slices from middle region, even if they have the same thickness. For this reason, several slices from both ends of a 50-mm gel rod were discarded, and the inner 40 slices were weighed. From the average of three independent experiments, the standard deviation of slice weight was calculated to be 8.23%) 7.860/o, 6.840/o, and 5.64%, respectively, for 3.75%, 7.5%, 15%, and 30% gel rods. With spacer rings, washers, and screw bolts of various number and size, it was possible to slice a gel rod of various size into slices of various thickness. The present method of slicing gel rods has several characteristics, as follows: Slices are obtainable from gel rods of 3.75% to 30% acrylamide concentration. It is not necessary to freeze gels prior to slicing. The time required for operation is relatively short. A gel slice can be separated from the blade assembly without touching it directly, because it is held inside a ring. We have been obtaining reproducible results using hand-made slicers of this type for several years. ACKNOWLEDGMENTS We express our thanks to Mr. and Mrs. S. Nitta, for their technical assistance.
Institute
of Medical
Science,
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
ORNSTEIN, L. (1964) Ann. N. Y. Acad. Sci. 121, 321. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404. RAYMOND, S. (1964) Ann. N. Y. Acud. Sci. 121, 350. CHRAMBACH, A. (1966) Anal. B&hem. 15,544. HOWE, C., LEE, L. T., AND OTTERSEN, 0. (1969) Appl. Microbial. 17, 18.3. HUTCHISON, C. A., III, EDGELL, M. H., AND SINSHEIMER. R. L. (1967) J. Mol. Biol. 23, 553. ARONSON, J. N., AND BORRIS, D. P. (1967) Anal. Biochem. 18, 27. BISHOP, D. H. L., CLAYBROOK, J. R., AND SPIEGELMAN, S. (1967) J. Mol. Biol. 26, 373. GRESSEL, J., AND WOLOWELSKY, J. (1968) Anal. Biochem. 22, 352. IANDOLO, J. J. (1970) Anal- Biochem. 36, 6.
SHORT
575
COMMUNICATIONS
11. SHEU, C. W., AND RIES, J. J. (1972) Anal. Biochem. 12. MAIZEL, J. V., JR. (1966) Science 151, 988. 13. GOLDBERGER. R. F. (1968) Anal. Biochem. 25, 46.
49,
TOSHIHARIHARUHIKO Department of Biophysics and Biochemistry Faculty of Science, University oj Tokyo Hongo, Tokyo 113, Japan Received September 26, 1972; accepted
July
23.
XIATSL-MCRA’ NODA
11, 1973
1 Present address: Department of Cancer Cell Research. Institute Science, Takanawa. Tokyo 108, Japan.
of Medical
56, 571-575 (1973)
BIOCHEMISTRY
SHORT
A Simple
Device
COMMUNICATIONAL
for
Transverse
of Polyacrylamide
Gel
Slicing
Rods
The use of electrophoresis on polyacrylamide gel has permitted remarkable resolutions of macromolecular mixtures (i-3). A number of successful methods to fractionate gel rods or gel slabs for enzymic assay and for radioactivity assay have been described. A slicer constructed with a series of wires, which resembles a cheese cutter, is suitable for slicing gels of medium acrylamide concentration, i.e., 5-15% (4). A bolted series of razor blades is adequate to slice slabs (5) and is also useful in slicing cylindrical gel rods (6) and square gel columns (7). Fractionation of a gel rod in a sequential fashion has been attained either with microtome-like slicers (8-11) or with gel extrusion system through a precision syringe and a fixed needle into a stream of eluting liquid (12). With improved microtomes Iandolo (10) and Sheu and Ries (11) have made it possible to section soft gels without freezing. Several difficulties, however, have arisen with these fractionation methods. With devices using wires or blades, distortion and breakage of gels often take place by the pressure on gels. Although this difficulty is partly reduced with the circular blade model of Goldberger (13)) it is required to freeze gels, especially of low acrylamide concentration, rock hard prior to slicing with these devices. In practice, however, the distortion of gels during freezing and the adherence of gel slices to cold met,al blades interfere. An automatic fractionation is possible with the gel extrusion system; however, this system may not be universally applicable because of the difficulty with hard gels. A difficulty with the sequential fractionation is that the fitness of a gel rod to the holder influences t’he precision of slicing (10, 11). In the present paper a method of gel slicing by the use of a razor blade assembly is described. This method overcomes some of these disadvantages mentioned above, because no freezing prior to slicing is required, gel slices are obtainable in a separate state from each other, and slices are obtainable from soft gels as well as from hard gels. 571 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
572
SHORT
COMMUNICATIONS
1. Photographs of gel slicers in assembly and in use: (a) A pair of bolts on a wooden block and razor blades mounted to form a razor blade assembly. Mounting spacer rings between the blades of the assembly. (c) A sample gel inserted in the holes of spacer rings. (d) Pushing the assembly down on the gel (e) Taking a ring out with a gel slice inside.
FIG.
put (h) rod rod.
A razor blade assembly was made as follows: Double-edge blades of stainless steel (Hi-Stainless, Feather Safety Razor Co., Osaka, Japan) were rinsed with ethyl acetate to remove wax, then with water, and dried. About 60 pieces of the blades, being separated from each other were connected with a pair of screw by 1 mm thick plastic washers, bolts and nuts. Use of a wooden block with two holes to support the
SHORT
COMMUNICATIONS
573
bolts in the vertical and parallel position helped this procedure (Fig. la). Fift,y plastic spacer rings, each of which was 1 mm thick and had a hole 6 mm in diameter, were put by the use of pincers into the gaps of the blades (Fig. lb). The spacer rings should be placed on the blade assembly so that. the hole of each ring might be free from t,he blade edge. The position of each ring on the blade assembly was adjusted by insertion of a plastic rod through the spacer rings so that a gel rod might be inserted smoothly through the holes. The assembly was again put on the wooden block, and a sample gel rod, 50 mm long and 5 mm in diameter, was inserted vertically into the core of the stack of spacer rings. The assembly should be wetted with water prior to inserting soft gels. No special lubricant was necessary for inserting rods. The fitness of a rod to the hole of spacer rings was loose. After a gel rod was inserted in the right position (Fi g. lc) , the tightness of connection of the blades should be controlled by driving the nuts in order that the spacer rings smoothly moved into the gaps. The rings with the gel rod inside the holes were pushed at once into the gaps of blades, until the holes disappeared into the gaps, while a part of each ring still appeared in the blades. This procedure was attained by turning the assembly upside down on the bench and pushing it by both ends of bolts (Fig. Id). Although the handling is not dangerous, it is preferable to cover the other side of blade edges by something like an aluminum plate, or a plastic sheet. After the pushing, each spacer ring with a sliced gel inside the hole was pulled out, by a pair of pincers (Fig. le), and the gel slice was transferred into a test tube. We separated a slice from a ring by the use of a needle or by immersing the ring and gel slice in a
574
SHORT
COMMUNICATIONS
small amount of buffer solution. The blade assembly was rinsed with water and dried for the next use. It took 5-10 min to slice a gel rod and to transfer slices into test tubes. For the determination of slicing precision, gel rods of 3.75% w/v acrylamide (0.1% w/v N,N’-methylenebisacrylamide), 7.5% (0.2%), 15% (0.4%) and 30% (0.8%) concentrations, respectively, were sliced. Every gel rod was sliced through the entire length. The slice weight, however, may not quantitatively represent the slice thickness for the few slices from both ends of a gel rod, because the terminal slice may be less thick t.han the distance between adjacent two blades, and because a gel rod often swells at the ends so that one or two slices from the end region may weigh more than the slices from middle region, even if they have the same thickness. For this reason, several slices from both ends of a 50-mm gel rod were discarded, and the inner 40 slices were weighed. From the average of three independent experiments, the standard deviation of slice weight was calculated to be 8.23%) 7.860/o, 6.840/o, and 5.64%, respectively, for 3.75%, 7.5%, 15%, and 30% gel rods. With spacer rings, washers, and screw bolts of various number and size, it was possible to slice a gel rod of various size into slices of various thickness. The present method of slicing gel rods has several characteristics, as follows: Slices are obtainable from gel rods of 3.75% to 30% acrylamide concentration. It is not necessary to freeze gels prior to slicing. The time required for operation is relatively short. A gel slice can be separated from the blade assembly without touching it directly, because it is held inside a ring. We have been obtaining reproducible results using hand-made slicers of this type for several years. ACKNOWLEDGMENTS We express our thanks to Mr. and Mrs. S. Nitta, for their technical assistance.
Institute
of Medical
Science,
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
ORNSTEIN, L. (1964) Ann. N. Y. Acad. Sci. 121, 321. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404. RAYMOND, S. (1964) Ann. N. Y. Acud. Sci. 121, 350. CHRAMBACH, A. (1966) Anal. B&hem. 15,544. HOWE, C., LEE, L. T., AND OTTERSEN, 0. (1969) Appl. Microbial. 17, 18.3. HUTCHISON, C. A., III, EDGELL, M. H., AND SINSHEIMER. R. L. (1967) J. Mol. Biol. 23, 553. ARONSON, J. N., AND BORRIS, D. P. (1967) Anal. Biochem. 18, 27. BISHOP, D. H. L., CLAYBROOK, J. R., AND SPIEGELMAN, S. (1967) J. Mol. Biol. 26, 373. GRESSEL, J., AND WOLOWELSKY, J. (1968) Anal. Biochem. 22, 352. IANDOLO, J. J. (1970) Anal- Biochem. 36, 6.
SHORT
575
COMMUNICATIONS
11. SHEU, C. W., AND RIES, J. J. (1972) Anal. Biochem. 12. MAIZEL, J. V., JR. (1966) Science 151, 988. 13. GOLDBERGER. R. F. (1968) Anal. Biochem. 25, 46.
49,
TOSHIHARIHARUHIKO Department of Biophysics and Biochemistry Faculty of Science, University oj Tokyo Hongo, Tokyo 113, Japan Received September 26, 1972; accepted
July
23.
XIATSL-MCRA’ NODA
11, 1973
1 Present address: Department of Cancer Cell Research. Institute Science, Takanawa. Tokyo 108, Japan.
of Medical