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A simple density-gradient engine for loading large-capacity zonal ultracentrifuge rotors
SHORT 4.
RUCHELMAN,
5.
RANDERATH,
6.
NEUHARD,
171
COMMUNICATIONS
M. W., J. Chem. Ed. 44, 110 (1967). K., AND RANDERATH, E., J. Chromatog. 22, 110 (1966). J., RANDERATH, E., AND RANDERATH, K., Anal. Biochem.
13, 211 (1965).
PATRICIA
J. T.
HASTINGS M’ONG
Department of Biochemistry University of Toronto Toronto, Canada Received August 10, 1967
A Simple
Density-Gradient
Large-Capacity
Zonal
Engine Ultracentrifuge
for
Loading Rotors
Many devices for preparing density gradients from sucrose solutions have been described (see, e.g., references l-5). These devices produce gradients of various profiles but are suitable only for the production of small volumes of gradient in tubes for use in swinging-bucket or anglehead rotors. The loading of the large-capacity, high-speed zonal ultracentrifuge rotors that have recently been developed (6) and are now beginning to come into general use presents some difficulties, since these rotors require a gradient engine which can form and pump gradients of volumes from 400-1200 ml in 30-60 min (7). One gradient engine with such capabilities is currently available (Beckman Instruments Inc., Palo Alto, Calif.) but, although it is very versatile, it is also expensive. This note describes a simple gradient engine that is readily constructed from commonly available apparatus and can be used to load zonal rotors with a variety of simple density gradients. A diagram of the apparatus is shown in Figure 1. The peristaltic pump should be capable of delivering at least 2 liters/hr against a back-pressure of 20 psi, and it must be possible to vary the flow rate without stopping the pump. A suitable pump is the Hughes Hiloflow (F. A. Hughes and Co., Epsom, Surrey, England), which was used throughout these experiments. The pressure gauge is calibrated for O-30 psi. The sucrose solutions, which are precooled to 4”, are kept cold by surrounding the two vessels, A and B, wit,h baths of ice. To form a gradient, the volume of heavy sucrose solution required is put in vessel A and the tube from A is filled with the sucrose solution to point C, at which point the tube is clamped. The volume of light
172
SHORT COMMUNICATIONS
sucrose solution required is put in the mixing vessel (B) and the apparatus is assembled, making sure that the ground-glass joint D is airtight. Tubes E and F are clamped, the delivery tube is connected to the fluid seal leading to the wall of the rotor and the magnetic stirrer is started. The peristaltic pump is switched on and, simultaneously, the clamp at C is released. Since the volume of gradient solution formed is equal to the volume of heavy sucrose solution introduced into the mixing vessel, the whole gradient has been delivered to the rotor when vessel A is emptied (assuming that the volume of liquid in the tube from
FIG. 1. Diagram
of density-gradient
engine. For details, see text.
vessel B to the rotor is negligible). A ‘Lcushion” of heavy sucrose solution can then be loaded into the rotor by clamping the delivery tube at points H and G, unclamping tubes E and F, and continuing to pump until all air has been expelled from E to F. G is then unclamped and F reclamped, and the required volume of cushion solution is pumped into the rotor. The same arrangement can be used to displace the contents of the rotor after centrifugation has been completed. A series of gradient profiles produced by this apparatus is shown in Figure 2. The gradient profile obtained is dependent only on the concentrations of light and heavy sucrose solutions and the ratio of the volumes of light and heavy solutions, so that the shapes of both vessels A and B (Fig. 1) are immaterial. However, it is necessary to have
SHORT
173
COMMUNICATIONS
available several different sizes of mixing vessel, because the smoothest gradients are obtained when the depth of liquid in the mixing vessel is between 3 and 20 cm. For example, to take the two extreme cases shown in Figure 2, a 400 ml convex gradient (curve (a)) is formed from 400 ml of heavy sucrose solution and 100 ml of light sucrose solution, while a 1200 ml gradient approximately linear with volume (curve (e) ) requires 1200 ml of heavy sucrose solution and 2400 ml of light sucrose solution. Gradient profiles more complex than those shown in Figure 2 can readily be produced by, for example, increasing the concentration of the heavy sucrose solution after part of the gradient has been delivered to the
0
0.2
04
06
08
I IO
FIG. 2. Density-gradient profiles produced by gradient engine from 10% (w/w) and 60% (w/w) sucrose solutions. The volume of heavy sucrose solution was 400 ml and the ratios of the volumes of light and heavy solutions were (a) 0.25 (0); (b) The flow rate was maintained 0.5 (0); Cc) 0.75 (A) ; (d) 1.0 (A) ; and (e) 2.0 (0). at 20 ml/min, and 20 ml fractions were collected.
rotor. Both simple and complex gradients formed by this apparatus are readily reproducible. In replicate experiments, the concentration of sucrose varied by less than 2% (w/w) at the steepest part of a convex gradient while the variation was less than 0.5% (w/w) at the shallow part. This apparatus provides a simple way of rapidly forming and introducing large volumes of gradient solution into zonal ultracentrifuge rotors. It is easily programmed to deliver a variety of density gradients of the type which are most generally useful in density-gradient centrifugation (5, 8). In addition, construction of the gradient engine is both simple and inexpensive.
174
SHORT
COMMUNICATIONS
ACKNOWLEDGMENT We are grateful to Miss Sylvia Fox for helpful discussions and to Dr. R. J. C. Harris for reading the manuscript. REFERENCES 1. ANDERSON, N. G., in “Physical Techniques in Biological Research” (G. Oster and A. W. Pollister, eds.), Vol. 3, p. 299. Academic Press, New York, 1956. 2. BBAKICE, M. K., in “Advances in Virus Research” (K. M. Smith and M. A. Lauffer, eds.), Vol. 7, p. 193. Academic Press, New York, 1969. 3. SAMUEL, S., Anal. Biochem. 7, 164 (1964). 4. SAMIS, H. V., JR., Anal. Biochem. 15, 355 (1966). 5. NOLL, H., Nature (London) 215, 366 (1967). 6. Natl. Cancer Inst. Monograph 21, (1966). 7. BIRNIE, G. D., Lab. Practice 16, 795 (1967). 8. ANDERSON, N. G., Natl. Cancer Inst. Monograph 21, 9 (1966).
G. D. BIRNIE D. R. HARVEY Division
of Experimental
Zmperial Cancer Research
Biology Fund
and
Virology
Burtonhole Lane, Mill Hill, London, N.W. Y, England Received August lY, 1967
An Absorption
System
for Collecting
Dog
Respiratory
CO2
During metabolic studies in the dog with W-labeled compounds it became necessary to measure the radioactivity expired as 14C0,. However it was found that the respiratory CO, was not efficiently absorbed by bubbling the expired air through large volumes of ethanolamine or sodium hydroxide. As this was probably due to the high air flow used and the consequent poor surface area of contact for absorption, we have designed an apparatus to overcome this difficulty. The principle is based on that used in the Nilcox absorption towers (Jencons (Scientific) Ltd., Mark Road, Hemel Hempstead, England) in which air is bubbled into a continuously circulating absorption solution in glass spiral tubing. The absorption system (see Fig. 1) consists of a 10 liter glass aspirator bottle containing 5 liters of ethanolamine (50% v/v) around which is coiled 7 meters of No. 26 Portex translucent vinyl tubing. The latter is connected to the base outlet of the aspirator and to the metabolism cage
RUCHELMAN,
5.
RANDERATH,
6.
NEUHARD,
171
COMMUNICATIONS
M. W., J. Chem. Ed. 44, 110 (1967). K., AND RANDERATH, E., J. Chromatog. 22, 110 (1966). J., RANDERATH, E., AND RANDERATH, K., Anal. Biochem.
13, 211 (1965).
PATRICIA
J. T.
HASTINGS M’ONG
Department of Biochemistry University of Toronto Toronto, Canada Received August 10, 1967
A Simple
Density-Gradient
Large-Capacity
Zonal
Engine Ultracentrifuge
for
Loading Rotors
Many devices for preparing density gradients from sucrose solutions have been described (see, e.g., references l-5). These devices produce gradients of various profiles but are suitable only for the production of small volumes of gradient in tubes for use in swinging-bucket or anglehead rotors. The loading of the large-capacity, high-speed zonal ultracentrifuge rotors that have recently been developed (6) and are now beginning to come into general use presents some difficulties, since these rotors require a gradient engine which can form and pump gradients of volumes from 400-1200 ml in 30-60 min (7). One gradient engine with such capabilities is currently available (Beckman Instruments Inc., Palo Alto, Calif.) but, although it is very versatile, it is also expensive. This note describes a simple gradient engine that is readily constructed from commonly available apparatus and can be used to load zonal rotors with a variety of simple density gradients. A diagram of the apparatus is shown in Figure 1. The peristaltic pump should be capable of delivering at least 2 liters/hr against a back-pressure of 20 psi, and it must be possible to vary the flow rate without stopping the pump. A suitable pump is the Hughes Hiloflow (F. A. Hughes and Co., Epsom, Surrey, England), which was used throughout these experiments. The pressure gauge is calibrated for O-30 psi. The sucrose solutions, which are precooled to 4”, are kept cold by surrounding the two vessels, A and B, wit,h baths of ice. To form a gradient, the volume of heavy sucrose solution required is put in vessel A and the tube from A is filled with the sucrose solution to point C, at which point the tube is clamped. The volume of light
172
SHORT COMMUNICATIONS
sucrose solution required is put in the mixing vessel (B) and the apparatus is assembled, making sure that the ground-glass joint D is airtight. Tubes E and F are clamped, the delivery tube is connected to the fluid seal leading to the wall of the rotor and the magnetic stirrer is started. The peristaltic pump is switched on and, simultaneously, the clamp at C is released. Since the volume of gradient solution formed is equal to the volume of heavy sucrose solution introduced into the mixing vessel, the whole gradient has been delivered to the rotor when vessel A is emptied (assuming that the volume of liquid in the tube from
FIG. 1. Diagram
of density-gradient
engine. For details, see text.
vessel B to the rotor is negligible). A ‘Lcushion” of heavy sucrose solution can then be loaded into the rotor by clamping the delivery tube at points H and G, unclamping tubes E and F, and continuing to pump until all air has been expelled from E to F. G is then unclamped and F reclamped, and the required volume of cushion solution is pumped into the rotor. The same arrangement can be used to displace the contents of the rotor after centrifugation has been completed. A series of gradient profiles produced by this apparatus is shown in Figure 2. The gradient profile obtained is dependent only on the concentrations of light and heavy sucrose solutions and the ratio of the volumes of light and heavy solutions, so that the shapes of both vessels A and B (Fig. 1) are immaterial. However, it is necessary to have
SHORT
173
COMMUNICATIONS
available several different sizes of mixing vessel, because the smoothest gradients are obtained when the depth of liquid in the mixing vessel is between 3 and 20 cm. For example, to take the two extreme cases shown in Figure 2, a 400 ml convex gradient (curve (a)) is formed from 400 ml of heavy sucrose solution and 100 ml of light sucrose solution, while a 1200 ml gradient approximately linear with volume (curve (e) ) requires 1200 ml of heavy sucrose solution and 2400 ml of light sucrose solution. Gradient profiles more complex than those shown in Figure 2 can readily be produced by, for example, increasing the concentration of the heavy sucrose solution after part of the gradient has been delivered to the
0
0.2
04
06
08
I IO
FIG. 2. Density-gradient profiles produced by gradient engine from 10% (w/w) and 60% (w/w) sucrose solutions. The volume of heavy sucrose solution was 400 ml and the ratios of the volumes of light and heavy solutions were (a) 0.25 (0); (b) The flow rate was maintained 0.5 (0); Cc) 0.75 (A) ; (d) 1.0 (A) ; and (e) 2.0 (0). at 20 ml/min, and 20 ml fractions were collected.
rotor. Both simple and complex gradients formed by this apparatus are readily reproducible. In replicate experiments, the concentration of sucrose varied by less than 2% (w/w) at the steepest part of a convex gradient while the variation was less than 0.5% (w/w) at the shallow part. This apparatus provides a simple way of rapidly forming and introducing large volumes of gradient solution into zonal ultracentrifuge rotors. It is easily programmed to deliver a variety of density gradients of the type which are most generally useful in density-gradient centrifugation (5, 8). In addition, construction of the gradient engine is both simple and inexpensive.
174
SHORT
COMMUNICATIONS
ACKNOWLEDGMENT We are grateful to Miss Sylvia Fox for helpful discussions and to Dr. R. J. C. Harris for reading the manuscript. REFERENCES 1. ANDERSON, N. G., in “Physical Techniques in Biological Research” (G. Oster and A. W. Pollister, eds.), Vol. 3, p. 299. Academic Press, New York, 1956. 2. BBAKICE, M. K., in “Advances in Virus Research” (K. M. Smith and M. A. Lauffer, eds.), Vol. 7, p. 193. Academic Press, New York, 1969. 3. SAMUEL, S., Anal. Biochem. 7, 164 (1964). 4. SAMIS, H. V., JR., Anal. Biochem. 15, 355 (1966). 5. NOLL, H., Nature (London) 215, 366 (1967). 6. Natl. Cancer Inst. Monograph 21, (1966). 7. BIRNIE, G. D., Lab. Practice 16, 795 (1967). 8. ANDERSON, N. G., Natl. Cancer Inst. Monograph 21, 9 (1966).
G. D. BIRNIE D. R. HARVEY Division
of Experimental
Zmperial Cancer Research
Biology Fund
and
Virology
Burtonhole Lane, Mill Hill, London, N.W. Y, England Received August lY, 1967
An Absorption
System
for Collecting
Dog
Respiratory
CO2
During metabolic studies in the dog with W-labeled compounds it became necessary to measure the radioactivity expired as 14C0,. However it was found that the respiratory CO, was not efficiently absorbed by bubbling the expired air through large volumes of ethanolamine or sodium hydroxide. As this was probably due to the high air flow used and the consequent poor surface area of contact for absorption, we have designed an apparatus to overcome this difficulty. The principle is based on that used in the Nilcox absorption towers (Jencons (Scientific) Ltd., Mark Road, Hemel Hempstead, England) in which air is bubbled into a continuously circulating absorption solution in glass spiral tubing. The absorption system (see Fig. 1) consists of a 10 liter glass aspirator bottle containing 5 liters of ethanolamine (50% v/v) around which is coiled 7 meters of No. 26 Portex translucent vinyl tubing. The latter is connected to the base outlet of the aspirator and to the metabolism cage