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An easily constructed, inexpensive, tube-puncturing device for density gradient centrifugation
ANALYTICAL
An
25, 252-256
BIOCHEMISTRY
Easily Device
Constructed, for
JOEL OPPENHEIM, Department Stritch
(1968)
inexpensive,
Density JULIAN
Gradient
Tube-Puncturing Centrifugation
SCHEINBUKS,
AND
of Microbiology, Loyola University School of Medicine, Hines, Illinois
Received February
LEON MARCUS (Chicago),
GO141
19, 1968
The excellent resolution of particles or molecules of differing molecular weight afforded by density gradient centrifugation has made this the method of choice for a variety of applications during the last decade (l-4). Concomitant with the ever-increasing interest in density gradient centrifugation as an analytical and preparative tool has has been an increase in the number of devices for tube puncture and fraction collection (5-16). High among the obvious requirements of such devices are structural rigidity and provision for constant temperature so that the gradient will not be disturbed by tube movement or convection due to temperature change during outflow of the contents of the tube. Lesser, but still important factors, are cost and ease of construction. In the present paper we describe an inexpensive, easily constructed, tube-puncturing device that has given trouble-free service in this and other laboratories for over three years. This device was designed for the IEC SB 110 swingingbucket rotor containing 40 ml polyallomer tubes. However, previous designs requiring slightly different dimensions have been used for tubes fitting the Spinco SW 25 rotor series. APPllRATUS Figure 1 shows a cross-section of the apparatus. The device consists of tube-puncturing and positioning and cooling sections. COiSSTRUCTIOX (a) The tube puncturing section. il2.9 cm diameter hole is drilled halfway through the center of a 5 X 5 in. supporting board of 112in. plywood. A 2 cm diameter hole is drilled through the center of the underside of the board into which is snugly fitted a 1 cm section of the threaded base of a cut-down, plast,ic, 2 cm diameter Vacutainer holder (Becton-Dickinson ‘Research Career Development Institutes of Health.
Awardee 252
l-K3-GM-31,
792-02, of the National
TUBE-PUNCTURING
253
DEVICE
B302.5). A 2.5 cm diameter rubber cushion (IEC 571) is glued into the opening on the upper side of the board which positions the cushion directly above the Vacutainer barrel. A double-ended needle may then be screwed into the base of the holder through the rubber cushion. (b) Positioning and cooling sectiojz. The density gradient tube is supported by a water-jacketed cylinder. The cylinder itself is a 50 ml polycarbonate centrifuge tube (IEC 1650) from which 1.5 cm of the lower portion has been removed. A 3 cm diameter hole is drilled through the center of the base of a 250 ml centrifuge bottle (IEC 2936) after 4 cm
SPRING CLAMP BOTTLElIEC 2936) 3
-COOLANT
INLET
DOUBLE-END 20G VACUTAINER NEEDLE FIG.
1. Schematic
representation
of tube-puncturing
device.
has been removed off the top of the bottle. The polycarbonate cylinder is then glued into the aperture at the base of the centrifuge bottle with a silicone rubber adhesive.2 The inlet and outlet. ports for coolant were formed by tightly wedging two plastic connectors (Be1 Art 1971) into holes drilled at the side of the bottle. All joints and connections were sealed watertight.? The water-jacketed section of the device is held in place over the puncturing section by two spring clamps fitted through wooden supports built into the sides of the base. The shanks of these ring clamps are fastened to the support frame. A steel extension ring with a 15 cm shank is fastened to the underside of the base with horseshoe staples. This in turn is clamped into the rigid support frame. (c) Tube puncture. After centrifugation, the gradient tube is placed into the tube holder and a vacuum sealing cap (IEC 1580) positioned * Dow
Corning
Lab
RTV
silastic
silicone
rubber
adhesive.
254
OPPENHEIM,
SCHEINBUKS,
AND
MARCUS
over the tube. The sole function of the sealing cap is to position the gradient tube in the holding cylinder and allow the operator to press the gradient tube firmly against the rubber cushion during tube puncture. It is not used to create a closed system for pumping nor is it tightened as per its usual function. The act of tightening the gasket, etc. would in itself disrupt the gradient. The gradient tube is punctured by screwing the Vacutainer needle into its base. Since the needle is screwed into the bottom of the polyallomer tube, no plug forms in the needle to retard the flow of the gradient,. EXAMPLE
OF
OPERATION
Recent studies in our laboratory have been concerned with the isolation of discrete polyribosome size classes from Amtobacter vinehndii (17-20). To achieve resolution and separation, extracts from exponentially growing Azotobacter cultures were obtained by osmotic lysis (19-21) and centrifuged through 40 ml of a 1037% exponential sucrose density gradient in an IEC SB 110 swinging-bucket rotor in an IEC B-35 ultracentrifuge at 105,000 g for 4 hr (19,20). After centrifugation, the polyallomer tubes were punctured as described above and the contents of the tubes allowed to drain through a specially constructed flow cell (22) flowing from top to bottom. The flow cell and tubing were prefilled with 40% sucrose in buffer to preclude convection and mixing during outflow. Absorbancy of the effluent was monitored continuously at 260 rnp during flow through a Beckman DB spectrophotometer connected to a Sargent SRL recorder for display (Fig. 2) (17, 18). Forty milliters of sucrose gradient fluid drains by gravity in
FIG.
2. Schematic
representation
of components
of the
analysis
system.
TCBE-PI’NCTURING
255
DEVICE
about, IO min. The flow rate is remarkably constant even though the gradient varies from 10 to 37% sucrose, Figure 3 shows an absorbancy trace of a typical experiment.. Seven to eight polyribosomal size classes in addition to the single ribosomes and ribosomal subunits are resolved routinely. A large region of heavy unresolved polyribosomre sediment, ahead of the fastest resolvable peak (19, 20). 20. A260 15-j
LO.-
0.5 -: b _ OT FIG. batter
3. Density winelan&.
B
gradient profile of ribosomal The bottom of the t.ube (B)
content of an extract represents the most
from dense
Azotoportion
of the gradient. COMMENTS
The gradient tube-puncturing device described in this paper aids greatly in the achievement of the degree of resolution and separation of the polyribosomes afforded by the excellent resolving power of sucrose density gradient centrifugation techniques. The device is an integral part of our postcentrifugation analysis system. The tube-puncturing device is readily constructed from general laboratory materials at a fraction of the cost of commercially available equipment. During three years of use in this laboratory this device has been found to be easily operated with a high degree of reliabi1it.y. With this apparatus and the polyallomer tubes, we have never experienced leakage of tube contents after puncture. ACXNOWLEDGMENTS This GM
u-ork was supported 14117 and National
in part Institutes
by U. S. Public Health of Heakh General
Servicr Research
Researrh Support
Grants Grant
256 FR 05368 to the to Carol Conzevoy
OPPEKHEIM,
Graduate Marcus
SCHEIiXBUKS,
Division of Loyola for the preparation
AXD
MARCUS
University (Chicago). of the figures.
We are grateful
REFERENCES C., BERTIIET, J., AND BE.IUFAT, H., Progr. Biophys. Biophys. Chem. 9, 325 ( 1959). 2. BRXKE, M. Ii., Bduan. Vi~zts Res. 7, 193 (1960). 3. VINOGRAD, J., AND HEARST, J. E., Prgr. Che,m. Org. Xat. Products 20, 372 (1962). 4. DEDUVE, C.: Harvey Lectures 59, 49 (1965). 5. BRITTEN, R. J., AND ROBERTS, R. B., S&ewe 131, 32 (1960). 6. HECKLY, R. J., Anal. Biochem. 1, 97 (1960). 7. SZYBALSKI, W., Experientia 16, 164 (1960). S. MARTIN, F. G., AND AMES, B. N.. J. Biol. Chem. 236, 1372 (1961). 9. BR.~KKE, M. K., il&. Biochem. 5,271 (1963). 10. DRESDEN, M., .\ND HoAGLAND, M. B., Science 149, 647 (1965). 11. SALO, T., Anal. Biochem. 10, 344 (1965). 12. NEWTON, IN., Anal. Biochem. 10, 361 (1965). 13. RON, E. Z., ROHLER, R. E., AND DAVIS, B. D., Science 153, 1119 (1966). 14. WATKINS, J., ASD TEE, D. E. H., Sci. Tools 13, 7 (1966). 15. COLEMAN, G., .~ND SYKES, J., sci. Tools 13, 43 (1966). 16. ROMANI, R. J., IND FISHER. 1,. Ei., Anal. Biochem. 21, 333 (1967). 17. MARCUS, L., RIS, H., H.~LVORSON, H. 0.. BHETTHAUER, R. K., AND BOCK, R. M., J. Cell Biol. 34, 510 (1967). 18. MARCUS, L., ASD H.~LVORSON, H. O., i/~ “The Nucleic Acids,” Vol. 12 of “Methods Enzymology” (I,. Grossman and K. Moldavc. eds.), art. 59, Academic Press, New York, 1968. 19. OPPENHEIM, J., AND M.~RCUS, L., Bacterial. Proc. 67, 120 (1967). 20. OPPENHEIM, J., SCHEINBUKS. J., Br~v.4, C.. AND M.sncus, L., Biochim. Biophys. Acta 161, 386 (1968). 21. ROBRISH, S. A., AND MARH, A. G., J. Buctr~~iol. 83, 100 (1962). 22. M~ICUS, L., AND BRUENISQ. CT., in manuscript (1963). 1. DEDUVE,
An
25, 252-256
BIOCHEMISTRY
Easily Device
Constructed, for
JOEL OPPENHEIM, Department Stritch
(1968)
inexpensive,
Density JULIAN
Gradient
Tube-Puncturing Centrifugation
SCHEINBUKS,
AND
of Microbiology, Loyola University School of Medicine, Hines, Illinois
Received February
LEON MARCUS (Chicago),
GO141
19, 1968
The excellent resolution of particles or molecules of differing molecular weight afforded by density gradient centrifugation has made this the method of choice for a variety of applications during the last decade (l-4). Concomitant with the ever-increasing interest in density gradient centrifugation as an analytical and preparative tool has has been an increase in the number of devices for tube puncture and fraction collection (5-16). High among the obvious requirements of such devices are structural rigidity and provision for constant temperature so that the gradient will not be disturbed by tube movement or convection due to temperature change during outflow of the contents of the tube. Lesser, but still important factors, are cost and ease of construction. In the present paper we describe an inexpensive, easily constructed, tube-puncturing device that has given trouble-free service in this and other laboratories for over three years. This device was designed for the IEC SB 110 swingingbucket rotor containing 40 ml polyallomer tubes. However, previous designs requiring slightly different dimensions have been used for tubes fitting the Spinco SW 25 rotor series. APPllRATUS Figure 1 shows a cross-section of the apparatus. The device consists of tube-puncturing and positioning and cooling sections. COiSSTRUCTIOX (a) The tube puncturing section. il2.9 cm diameter hole is drilled halfway through the center of a 5 X 5 in. supporting board of 112in. plywood. A 2 cm diameter hole is drilled through the center of the underside of the board into which is snugly fitted a 1 cm section of the threaded base of a cut-down, plast,ic, 2 cm diameter Vacutainer holder (Becton-Dickinson ‘Research Career Development Institutes of Health.
Awardee 252
l-K3-GM-31,
792-02, of the National
TUBE-PUNCTURING
253
DEVICE
B302.5). A 2.5 cm diameter rubber cushion (IEC 571) is glued into the opening on the upper side of the board which positions the cushion directly above the Vacutainer barrel. A double-ended needle may then be screwed into the base of the holder through the rubber cushion. (b) Positioning and cooling sectiojz. The density gradient tube is supported by a water-jacketed cylinder. The cylinder itself is a 50 ml polycarbonate centrifuge tube (IEC 1650) from which 1.5 cm of the lower portion has been removed. A 3 cm diameter hole is drilled through the center of the base of a 250 ml centrifuge bottle (IEC 2936) after 4 cm
SPRING CLAMP BOTTLElIEC 2936) 3
-COOLANT
INLET
DOUBLE-END 20G VACUTAINER NEEDLE FIG.
1. Schematic
representation
of tube-puncturing
device.
has been removed off the top of the bottle. The polycarbonate cylinder is then glued into the aperture at the base of the centrifuge bottle with a silicone rubber adhesive.2 The inlet and outlet. ports for coolant were formed by tightly wedging two plastic connectors (Be1 Art 1971) into holes drilled at the side of the bottle. All joints and connections were sealed watertight.? The water-jacketed section of the device is held in place over the puncturing section by two spring clamps fitted through wooden supports built into the sides of the base. The shanks of these ring clamps are fastened to the support frame. A steel extension ring with a 15 cm shank is fastened to the underside of the base with horseshoe staples. This in turn is clamped into the rigid support frame. (c) Tube puncture. After centrifugation, the gradient tube is placed into the tube holder and a vacuum sealing cap (IEC 1580) positioned * Dow
Corning
Lab
RTV
silastic
silicone
rubber
adhesive.
254
OPPENHEIM,
SCHEINBUKS,
AND
MARCUS
over the tube. The sole function of the sealing cap is to position the gradient tube in the holding cylinder and allow the operator to press the gradient tube firmly against the rubber cushion during tube puncture. It is not used to create a closed system for pumping nor is it tightened as per its usual function. The act of tightening the gasket, etc. would in itself disrupt the gradient. The gradient tube is punctured by screwing the Vacutainer needle into its base. Since the needle is screwed into the bottom of the polyallomer tube, no plug forms in the needle to retard the flow of the gradient,. EXAMPLE
OF
OPERATION
Recent studies in our laboratory have been concerned with the isolation of discrete polyribosome size classes from Amtobacter vinehndii (17-20). To achieve resolution and separation, extracts from exponentially growing Azotobacter cultures were obtained by osmotic lysis (19-21) and centrifuged through 40 ml of a 1037% exponential sucrose density gradient in an IEC SB 110 swinging-bucket rotor in an IEC B-35 ultracentrifuge at 105,000 g for 4 hr (19,20). After centrifugation, the polyallomer tubes were punctured as described above and the contents of the tubes allowed to drain through a specially constructed flow cell (22) flowing from top to bottom. The flow cell and tubing were prefilled with 40% sucrose in buffer to preclude convection and mixing during outflow. Absorbancy of the effluent was monitored continuously at 260 rnp during flow through a Beckman DB spectrophotometer connected to a Sargent SRL recorder for display (Fig. 2) (17, 18). Forty milliters of sucrose gradient fluid drains by gravity in
FIG.
2. Schematic
representation
of components
of the
analysis
system.
TCBE-PI’NCTURING
255
DEVICE
about, IO min. The flow rate is remarkably constant even though the gradient varies from 10 to 37% sucrose, Figure 3 shows an absorbancy trace of a typical experiment.. Seven to eight polyribosomal size classes in addition to the single ribosomes and ribosomal subunits are resolved routinely. A large region of heavy unresolved polyribosomre sediment, ahead of the fastest resolvable peak (19, 20). 20. A260 15-j
LO.-
0.5 -: b _ OT FIG. batter
3. Density winelan&.
B
gradient profile of ribosomal The bottom of the t.ube (B)
content of an extract represents the most
from dense
Azotoportion
of the gradient. COMMENTS
The gradient tube-puncturing device described in this paper aids greatly in the achievement of the degree of resolution and separation of the polyribosomes afforded by the excellent resolving power of sucrose density gradient centrifugation techniques. The device is an integral part of our postcentrifugation analysis system. The tube-puncturing device is readily constructed from general laboratory materials at a fraction of the cost of commercially available equipment. During three years of use in this laboratory this device has been found to be easily operated with a high degree of reliabi1it.y. With this apparatus and the polyallomer tubes, we have never experienced leakage of tube contents after puncture. ACXNOWLEDGMENTS This GM
u-ork was supported 14117 and National
in part Institutes
by U. S. Public Health of Heakh General
Servicr Research
Researrh Support
Grants Grant
256 FR 05368 to the to Carol Conzevoy
OPPEKHEIM,
Graduate Marcus
SCHEIiXBUKS,
Division of Loyola for the preparation
AXD
MARCUS
University (Chicago). of the figures.
We are grateful
REFERENCES C., BERTIIET, J., AND BE.IUFAT, H., Progr. Biophys. Biophys. Chem. 9, 325 ( 1959). 2. BRXKE, M. Ii., Bduan. Vi~zts Res. 7, 193 (1960). 3. VINOGRAD, J., AND HEARST, J. E., Prgr. Che,m. Org. Xat. Products 20, 372 (1962). 4. DEDUVE, C.: Harvey Lectures 59, 49 (1965). 5. BRITTEN, R. J., AND ROBERTS, R. B., S&ewe 131, 32 (1960). 6. HECKLY, R. J., Anal. Biochem. 1, 97 (1960). 7. SZYBALSKI, W., Experientia 16, 164 (1960). S. MARTIN, F. G., AND AMES, B. N.. J. Biol. Chem. 236, 1372 (1961). 9. BR.~KKE, M. K., il&. Biochem. 5,271 (1963). 10. DRESDEN, M., .\ND HoAGLAND, M. B., Science 149, 647 (1965). 11. SALO, T., Anal. Biochem. 10, 344 (1965). 12. NEWTON, IN., Anal. Biochem. 10, 361 (1965). 13. RON, E. Z., ROHLER, R. E., AND DAVIS, B. D., Science 153, 1119 (1966). 14. WATKINS, J., ASD TEE, D. E. H., Sci. Tools 13, 7 (1966). 15. COLEMAN, G., .~ND SYKES, J., sci. Tools 13, 43 (1966). 16. ROMANI, R. J., IND FISHER. 1,. Ei., Anal. Biochem. 21, 333 (1967). 17. MARCUS, L., RIS, H., H.~LVORSON, H. 0.. BHETTHAUER, R. K., AND BOCK, R. M., J. Cell Biol. 34, 510 (1967). 18. MARCUS, L., ASD H.~LVORSON, H. O., i/~ “The Nucleic Acids,” Vol. 12 of “Methods Enzymology” (I,. Grossman and K. Moldavc. eds.), art. 59, Academic Press, New York, 1968. 19. OPPENHEIM, J., AND M.~RCUS, L., Bacterial. Proc. 67, 120 (1967). 20. OPPENHEIM, J., SCHEINBUKS. J., Br~v.4, C.. AND M.sncus, L., Biochim. Biophys. Acta 161, 386 (1968). 21. ROBRISH, S. A., AND MARH, A. G., J. Buctr~~iol. 83, 100 (1962). 22. M~ICUS, L., AND BRUENISQ. CT., in manuscript (1963). 1. DEDUVE,