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Two swinging-bucket ultracentrifugal filters for receptor-binding assays
ANALYTICAL
BIOCHEMISTRY
105,
268-273 (1980)
Two Swinging-Bucket Ultracentrifugal for Receptor-Binding Assays
Filters
ROBERT FREUNDLICH AND DERMOT B. TAYLOR Department
of Pharmacology
and Brain Research Institute, Los Angeles, California 90024
UCLA
School
of Medicine,
Received November 14, 1979 Centrifugal filters for SW 25.1 and 50.1 swinging-bucket ultracentrifuge rotors have been tested up to the maximum speeds allowed, 90,000 and 3OO,OOOg,respectively. The filters are. 1 and 0.5 in. in diameter and accept standard 25mm polycarbonate filter membranes. The filter membranes are both cup-shaped to prevent loss of particulates to the support materials of the filters. The filter for the SW 25.1 rotor can take 0.75 ml and that of the 50.1 head 0.5 ml. The fluid retained after centrifuging consists of the fluid on the filter membrane and in its pores and that retained by the material filtered. The calculated volume of the pores of the 0.2~pm filter was 0.46 ~1. Total liquid retentions of about 0.8 ~1 have been achieved with both filters using a particulate concentration of 0.5 mg/ml.
Receptor-binding assays require the independent accurate determination of the free and bound ligand on the same preparation. To do this several equilibrium and nonequilibrium methods for separating the free and bound ligand have been developed and reviewed (1). Washing techniques by filtration or centrifugation are only useful when bound ligand loss in the wash is negligible and this is not always easy to prove experimentally or justify theoretically. Because of these and other complications involved in ion-exchange-controlled ligand binding (2,3), effort has been devoted to the separation of bound and free ligand by filtration produced by suction and by ultracentrifugation. In our previous method for the receptorbinding assay (4), a suspension of receptor particles was filtered on a 0.2~pm Nucleopore membrane in the shape of an inverted truncated cone held in place by suction. Membrane particles have the ability to cling loosely to the surfaces of containers as fluid level drops and the shape of the filter precludes loss of material to the membrane 0003-2697/80/100268-06$02.00/O Copyright All rights
0 1960 by Academic Press, Inc. of reproduction in any form reserved.
268
supports. The particulates were estimated by difference weighing and the radiolabeled ligand by scintillation counting of the airdried membrane. The results had a relative standard deviation of 2-3%. The liquid retained on the membranes was 3-5 ~1 and this did not interfere with the assay because the values were obtained from the difference between the individuals of a pair of filter membranes treated identically except that one contained an agent that specifically blocked the receptors. In order to improve the method and especially to reduce the amount of fluid left on the filter membrane, two filters have been tested for the SW 25.1 and SW 50.1 rotors of a Beckman Model L ultracentrifuge. MATERIALS
AND METHODS
The Filter for the SW 25.1 Rotor
The filter unit (Fig. IA) accepts a 25mm Nucleopore filter membrane and was machined from a slab of optical-quality (no discontinuities) polycarbonate. The spherical end of the filter body had a radius (13 .O mm)
ULTRACENTRIFUGAL
RECEPTOR
269
FILTERS
/PLUNGER
&U-p.
E4kEiANE 134 FILTER FORMING RING
PLUNGER GUIDE MEMBRANE FILTER FORMING RING
SLOTTED FILTER PLATE
FILTER HOLDER
,’
,I .’
tl2-7
,’
,’
,’ ,’
,’
,,:‘I ,’ ,’ I 8, 1 /’ ; ’ 0 ( : ; : ’ I
0
A
FIG. 1. Section through l-in. filter for the SW 25.1 rotor. (A) On the left is an expanded view showing the membrane-retaining collar which is put in place after the plunger guide has been removed. On the right, the filter is shown with the unformed membrane in place and the plunger and plunger guide. The tilter holder holds the plunger guide so that it does not press on the filter membrane. (B) A portion of the castellated membrane-forming ring for the SW 25.1 rotor filter. At the left is a section through the ring showing the curvature of the spherical surface and the bottom (dashed line) of one of the grooves. The center diagram shows the profile of one of the 24 membrane-accommodating grooves. Each groove has an angle of 124.7” at the periphery and comes to a point at the inner edge of the ring.
a little greater than the radius of the inside of the centrifuge bucket. Thus the contact between the filter and the bucket was ringshaped and was not the tip of the filter body. Hence high “g” caused compression of the filter rather than expansion and prevented incipient failure seen as hair cracks under dark field illumination. The filters tended to remain stuck in the bucket after a run. The problem was relieved by removing a little plastic at the junction of the hemispherical end and the cylindrical part of the body. The filter plate was a finely slotted polycarbonate filter disk from a General Electric 25mm Swin-Lok filtration holder. Two designs of polycarbonate membrane-forming rings have been used (Figs. 1A and B). In Fig. lA, the cross section of the ring is
a 45” isosceles triangle and the ring is smooth. When the polycarbonate membrane is pushed into place with the plunger, the bottom part of the membrane stays flat, but the sides become corrugated and if the plunger is removed, the membrane springs back. To prevent this the membrane-retaining collar (Fig. 1A) is dropped into the cavity to hold the membrane. Within 20 to 30 s after the liquid is placed in the cup-shaped membrane, a film of liquid forms between the membrane corrugations and the support ring. The film now holds the membrane and the retaining collar can be removed. The filter is immediately centrifuged at low speed (5000 rpm for about 2 min) until the liquid filters through. The speed is then raised to 25,000 rpm. If filtration is rapid, it is not necessary to use
270
FREUNDLICH
AND TAYLOR
1
a low speed first as all the liquid gets through before the highest “g” values are attained. During the run, the filter membrane becomes creased against the forming ring and ,PLUNGER it also tends to slip down the ramp of the forming ring to form small circular creases at the line where the inner edge of the ring rests on the plate. Since creases and folds in the membrane contribute to liquid retention, an improved support ring was designed. The modified ring (Fig. 1B) was made from the ring described above, but had 24 V-shaped grooves in its upper surface. The folding of the membrane on the original smooth membrane-forming rings is caused by the circumference of the membrane being 9- 11 CENTRIFUGE TUBE mm greater than the outside circumference FILTER PLATE of the forming ring. The grooves increased INSERT the circumference of the ring so that the MENISCUS EOUALIZING PORE membrane would be pressed into them withANTI-CAPILLARITY , 1 cm , out folding. The upper surface of the ring ANNULUS was also cut to be part of the surface of a sphere so that in section the surface went FIG. 2. The assembled filter for the SW 50.1 rotor is shown at the bottom with an expanded view of the through both inside apexes of the triangular cross section of the ring as shown in Fig. 1B. membrane-centering and -loading equipment above it. This reduced the tendency of the membrane to slide down the upper surface of the ring cavity partly empty. A pore through the insert at a low level allows the menisci in and form liquid-holding creases at the juncthe two spaces to equalize, but when the tion of the ring and the plate. centrifuge is stopped, capillarity caused fluid from the well to flow into the annulus and The 0.5-k. Filter for the SW 50.1 Rotor rise past the plate to the membrane. However, when the pore is 12.0 mm from the The filter (Fig. 2) is assembled in a 0.5in. polyallomer centrifuge tube and consists of bottom for the insert, both menisci are just a cup-shaped filter membrane resting on a below the pore and stay there when the centrifuge stops. The filter plate has a center filter plate supported by a specially designed and six small holes arranged hexagonally. polycarbonate insert. The insert provides space for the filtrate while ensuring that The rounded end of the insert has a radius of 6.18 mm, which is slightly larger than none of it can get back to the membrane when the centrifuge is stopped. When an that of the inside of the centrifuge tube (6.08 mm). This ensures compression stress insert is made a close fit for the centrifuge tube, fluid gets between the insert and the as in the case of the filter for the 25.1 rotor. To set up the litter, the membrane-centertube and can rise to the membrane by capillarity. To prevent this, the insert was pro- ing collar is fitted to the centrifuge tube and the membrane placed on it. After adding vided with a l-mm annulus on its outside. During centrifugation fluid enters both the the plunger guide, the plunger is used to press central cavity and the annulus, and tends to the membrane into place. Then the guide and collar are moved up the plunger, and the fill the annulus while leaving the central
271
ULTRACENTRIFUGALRECEFTORFILTERS
top part of the centrifuge tube around the membrane cup is rolled between the finger and thumb with considerable pressure to set the folds in the membrane. The plunger is removed, and the filter is ready to accept 0.5 ml of fluid. At 50,000 r-pm, the centrifugal force is 244,000g at the membrane. The plunger and plunger guide were made of Plexiglas, the membrane centering collar of Teflon, and the plate and insert of polycarbonate. Testing Procedures The test material used was a suspension of nicotinic acetylcholine receptor-bearing membrane fragments prepared as before (45). Protein was estimated spectroscopically (6). In determining the effect of centrifugal force on fluid retention, the filters were accelerated to the desired speed and held there for 1 min and the centrifuge was allowed to decelerate as usual. The holdup was determined by subtracting the mean weight of three air-dried filters using 10 mM 4morpholinepropanesulfonic acid buffer, pH 7.4, alone from the mean weight of three similar filters in which the buffer also contained 0.5 M KBr. RESULTS
The l-in. Filter in the SW 25.1 Rotor Using the original membrane support ring and a loading of 0.75 ml on each filter, liquid holdup was not significantly affected by prolonging the time at 25,000 rpm. The values were 1.36, 1.53, 1.58,and 1.60plat0,5, 15, and 30 min, respectively. The results for four different determinations of liquid holdup as a function of speed are shown in Fig. 3A. For the bottom curve, a filter loading of 0.5 ml was used while 0.75 ml was used for the other three. In all four experiments a mean standard deviation for each whole experiment was determined. The square points were determined using the original smooth membrane-forming ring. The points show greater scatter (SD
= 2.82%). The circles were determined using the castellated membrane-forming rings giving an overall standard deviation of 1.13%. The difference between the two standard deviations was significant. As mentioned earlier, at the higher speeds the membrane tends to slide down the surface of the membrane-shaping ring to form a circle of folds or creases at the bottom of the ring where it joins the plate. The process can be seen to start at opposite sides of the bottom of the cup where the slots in the filter plate are tangential to the circumference of the bottom of the cup. To try to prevent crease formation, two slots in the filter plate on each side were filled and machined flat. The results of this are shown by the crosses (Fig. 3A). This method tended to reduce crease formation and at the lower speeds made little difference to liquid holdup but at 20,000 and 25,000 rpm it increased liquid holdup. The bottom curve was obtained by loading 0.5 ml instead of 0.75 ml and immediately centrifuging the filters. The holdup was less presumably because the liquid did not have sufficient time to saturate all of the filter. All of the curves relating liquid holdup to speed are concave upward and appear to be approaching an asymptote although they are not simple exponentials. When the log of holdup was plotted against speed there was some residual concavity. Figure 4 shows that with up to 0.4 mg/filter or 0.53 mg/ml of original solution, the holdup is a linear function of the tissue load. Extrapolation of the least-squares regression line gives the holdup by the unloaded membrane as 0.96 ~1. An attempt to measure this directly by filtering 0.5 M KBr alone gave a value of 0.64 ~1, and the calculated volume of the pores in the membrane (see Discussion) was 0.46 ~1. The OS-in. Filter in the SW 50.1 Rotor The crosses (Fig. 3B) indicate ship between fluid holdup and the 0.5-in. filter in the 50.1 head. were obtained by using the filter
the relationspeed using The crosses as described
272
FREUNDLICH
AND TAYLOR
B
I 5
I 10
I 15
I 20 SPEED
I 25 IN THOUSANDS
I
I
I
I
I
10
20 OF RPM.
30
40
50
FIG. 3. The relationship between the amount of liquid retained on the filter membrane and the t-pm. (A) Using the SW 25.1 rotor (Fig. 1). An aliquot of 0.75 ml was placed on each filter except in the case of the lowest curve where it was 0.5 ml: 0, using the smooth membrane-forming ring; 0, using castellated ring; +, using castellated membrane-forming ring and partly filled-in support disk; A, using 0.5 ml and smooth membrane-forming ring. (B) +, using 0.5 ml and the SW 50.1 rotor; 0, same with a disk of polypropylene gauze between the filter membrane and the filter disk. The last points on each curve were associated with some collapse of three out of six of the centrifuge tubes in each case.
and 0.5 ml of fluid. The circles were obtained by the same technique except that there was a disk of polypropylene gauze between the filter membrane and the filter disk in each case in order to see if the liquid holdup could be decreased by reducing the area of contact between the filter membrane and its support. In fact, this had very little effect. The line in Fig. 3B has been drawn through the means of the first four points. The last two points were associated with partial collapse of half of the tubes in both cases. The collapse was associated with irregularities in centrifuge tube wall thickness in all cases. Up to 25,000 r-pm, the circles (Fig. 3A) using the l-in. filter fit the curve of Fig. 3B closely. Liquid Holdup Using the Suction Filtration Method
Using the 0.5 filtration method
M
KBr method the suction (4) (in triplicate) gave a
value of 2.96 ~1 using 0.75 ml of fluid and 2.45 ~1 using 0.5 ml of fluid. Soluble Protein in the Filtrates
Since it was possible that 0.5 M KBr might solubilize some of the insoluble protein, filtrates from 12 pooled filtrates from controls containing 0.5 M KBr were compared to similar pooled filtrates from controls containing no KBr. The KBr solution contained 0.24 mg/ml and that from the controls the same amount of soluble protein. DISCUSSION
When the buckets are opened after a run at 25,000 rpm, the membranes can be seen to be moist with more moisture present at the lower part of the membrane and especially where the membrane has been in contact with the grid of the filtration plate. The upper parts of the membrane are usually
ULTRACENTRIFUGAL
--A -6
I
)0
I
/
0.2
0.1 DRY
TISSUE
WEIGHT
I
0.3 PER
FILTER,
0.4 mq
FIG. 4. Relationship between the amount of liquid retained and the amount of particulate matter placed on the filter using the SW 25.1 rotor. The least-squares regression line cuts the vertical axis at 0.96 ~1. Point A represents the liquid retained when 0.75 ml of 0.5 M KBr was used containing no paniculates. Point B represents the calculated volume of liquid required to fill all the membrane pores.
relatively dry. It appears that there is residual fluid at least on some of the membrane surfaces, in some of its pores, and where the membrane is in contact with the supporting material of the filter itself. The 0.2-pm pore density of the lo-pmthick membranes is given as 3 x 10s crne2 (7) corresponding to a total pore volume of 0.46 @membrane. This accounts for about half of the experimentally determined holdup of 0.96 ~1 obtained by extrapolation of the data in Fig. 4. The somewhat lower value of 0.6 ~1 obtained by filtering 0.75 ml of 0.5 M KBr in 10 mM 4-morpholinepro-
RECEPTOR
FILTERS
273
panesulfonic acid buffer may have been due to incomplete wetting of the filter membrane. All the curves of holdup versus speed seem to be approaching an asymptote and this was particularly true in the case of the curve (crosses) in Fig. 3A where the area of contact between the filter membrane and the support disk had been increased. Some of the irreducible holdup must be due to surface imperfections in the machined surfaces of the filter. The situation might be improved by polishing the upper surface of the membrane-forming ring of the l-in. filter or the plate of the OS-in. filter. Thinner membranes with smaller pores are available, but have not been tried. Higher tissue concentrations in smaller volumes would improve the signal to noise ratio. A centrifuge rotor which goes up to 65,000 rpm with buckets taking 0.5-in. tubes is available. Treating all but a circle of 1 cm diameter of the 25-mm filter membranes with a plastic spray to fill the holes would allow adequate filtration, but would reduce holdup. REFERENCES 1. Bennett, J. P., Jr. (1978) in Neurotransmitter Receptor Binding (Yamamura, H. I., Enna, S. J., and Kuhar, M. J., eds.), pp. 57-90, Raven Press, New York. 2. Taylor, D. B. (1973)J. Pharmacol. Exp. Ther. 186, 537-551. 3. Helfferich, F. (1962) Ion Exchange, McGraw-Hill, New York. 4. Spivak, C. E., and Taylor, D. B. (1977) Anal. Biochem. 77, 274-279. 5. Franklin, F. I., and Potter, L. T. (1972) FEBS Letr. 28, 101. 6. Kalb, V. F., and Bernluhr, R. W. (1977) Anal. Biochem. 82,362-371. 7. General Electric Catalogue (1978) Lab 40.
BIOCHEMISTRY
105,
268-273 (1980)
Two Swinging-Bucket Ultracentrifugal for Receptor-Binding Assays
Filters
ROBERT FREUNDLICH AND DERMOT B. TAYLOR Department
of Pharmacology
and Brain Research Institute, Los Angeles, California 90024
UCLA
School
of Medicine,
Received November 14, 1979 Centrifugal filters for SW 25.1 and 50.1 swinging-bucket ultracentrifuge rotors have been tested up to the maximum speeds allowed, 90,000 and 3OO,OOOg,respectively. The filters are. 1 and 0.5 in. in diameter and accept standard 25mm polycarbonate filter membranes. The filter membranes are both cup-shaped to prevent loss of particulates to the support materials of the filters. The filter for the SW 25.1 rotor can take 0.75 ml and that of the 50.1 head 0.5 ml. The fluid retained after centrifuging consists of the fluid on the filter membrane and in its pores and that retained by the material filtered. The calculated volume of the pores of the 0.2~pm filter was 0.46 ~1. Total liquid retentions of about 0.8 ~1 have been achieved with both filters using a particulate concentration of 0.5 mg/ml.
Receptor-binding assays require the independent accurate determination of the free and bound ligand on the same preparation. To do this several equilibrium and nonequilibrium methods for separating the free and bound ligand have been developed and reviewed (1). Washing techniques by filtration or centrifugation are only useful when bound ligand loss in the wash is negligible and this is not always easy to prove experimentally or justify theoretically. Because of these and other complications involved in ion-exchange-controlled ligand binding (2,3), effort has been devoted to the separation of bound and free ligand by filtration produced by suction and by ultracentrifugation. In our previous method for the receptorbinding assay (4), a suspension of receptor particles was filtered on a 0.2~pm Nucleopore membrane in the shape of an inverted truncated cone held in place by suction. Membrane particles have the ability to cling loosely to the surfaces of containers as fluid level drops and the shape of the filter precludes loss of material to the membrane 0003-2697/80/100268-06$02.00/O Copyright All rights
0 1960 by Academic Press, Inc. of reproduction in any form reserved.
268
supports. The particulates were estimated by difference weighing and the radiolabeled ligand by scintillation counting of the airdried membrane. The results had a relative standard deviation of 2-3%. The liquid retained on the membranes was 3-5 ~1 and this did not interfere with the assay because the values were obtained from the difference between the individuals of a pair of filter membranes treated identically except that one contained an agent that specifically blocked the receptors. In order to improve the method and especially to reduce the amount of fluid left on the filter membrane, two filters have been tested for the SW 25.1 and SW 50.1 rotors of a Beckman Model L ultracentrifuge. MATERIALS
AND METHODS
The Filter for the SW 25.1 Rotor
The filter unit (Fig. IA) accepts a 25mm Nucleopore filter membrane and was machined from a slab of optical-quality (no discontinuities) polycarbonate. The spherical end of the filter body had a radius (13 .O mm)
ULTRACENTRIFUGAL
RECEPTOR
269
FILTERS
/PLUNGER
&U-p.
E4kEiANE 134 FILTER FORMING RING
PLUNGER GUIDE MEMBRANE FILTER FORMING RING
SLOTTED FILTER PLATE
FILTER HOLDER
,’
,I .’
tl2-7
,’
,’
,’ ,’
,’
,,:‘I ,’ ,’ I 8, 1 /’ ; ’ 0 ( : ; : ’ I
0
A
FIG. 1. Section through l-in. filter for the SW 25.1 rotor. (A) On the left is an expanded view showing the membrane-retaining collar which is put in place after the plunger guide has been removed. On the right, the filter is shown with the unformed membrane in place and the plunger and plunger guide. The tilter holder holds the plunger guide so that it does not press on the filter membrane. (B) A portion of the castellated membrane-forming ring for the SW 25.1 rotor filter. At the left is a section through the ring showing the curvature of the spherical surface and the bottom (dashed line) of one of the grooves. The center diagram shows the profile of one of the 24 membrane-accommodating grooves. Each groove has an angle of 124.7” at the periphery and comes to a point at the inner edge of the ring.
a little greater than the radius of the inside of the centrifuge bucket. Thus the contact between the filter and the bucket was ringshaped and was not the tip of the filter body. Hence high “g” caused compression of the filter rather than expansion and prevented incipient failure seen as hair cracks under dark field illumination. The filters tended to remain stuck in the bucket after a run. The problem was relieved by removing a little plastic at the junction of the hemispherical end and the cylindrical part of the body. The filter plate was a finely slotted polycarbonate filter disk from a General Electric 25mm Swin-Lok filtration holder. Two designs of polycarbonate membrane-forming rings have been used (Figs. 1A and B). In Fig. lA, the cross section of the ring is
a 45” isosceles triangle and the ring is smooth. When the polycarbonate membrane is pushed into place with the plunger, the bottom part of the membrane stays flat, but the sides become corrugated and if the plunger is removed, the membrane springs back. To prevent this the membrane-retaining collar (Fig. 1A) is dropped into the cavity to hold the membrane. Within 20 to 30 s after the liquid is placed in the cup-shaped membrane, a film of liquid forms between the membrane corrugations and the support ring. The film now holds the membrane and the retaining collar can be removed. The filter is immediately centrifuged at low speed (5000 rpm for about 2 min) until the liquid filters through. The speed is then raised to 25,000 rpm. If filtration is rapid, it is not necessary to use
270
FREUNDLICH
AND TAYLOR
1
a low speed first as all the liquid gets through before the highest “g” values are attained. During the run, the filter membrane becomes creased against the forming ring and ,PLUNGER it also tends to slip down the ramp of the forming ring to form small circular creases at the line where the inner edge of the ring rests on the plate. Since creases and folds in the membrane contribute to liquid retention, an improved support ring was designed. The modified ring (Fig. 1B) was made from the ring described above, but had 24 V-shaped grooves in its upper surface. The folding of the membrane on the original smooth membrane-forming rings is caused by the circumference of the membrane being 9- 11 CENTRIFUGE TUBE mm greater than the outside circumference FILTER PLATE of the forming ring. The grooves increased INSERT the circumference of the ring so that the MENISCUS EOUALIZING PORE membrane would be pressed into them withANTI-CAPILLARITY , 1 cm , out folding. The upper surface of the ring ANNULUS was also cut to be part of the surface of a sphere so that in section the surface went FIG. 2. The assembled filter for the SW 50.1 rotor is shown at the bottom with an expanded view of the through both inside apexes of the triangular cross section of the ring as shown in Fig. 1B. membrane-centering and -loading equipment above it. This reduced the tendency of the membrane to slide down the upper surface of the ring cavity partly empty. A pore through the insert at a low level allows the menisci in and form liquid-holding creases at the juncthe two spaces to equalize, but when the tion of the ring and the plate. centrifuge is stopped, capillarity caused fluid from the well to flow into the annulus and The 0.5-k. Filter for the SW 50.1 Rotor rise past the plate to the membrane. However, when the pore is 12.0 mm from the The filter (Fig. 2) is assembled in a 0.5in. polyallomer centrifuge tube and consists of bottom for the insert, both menisci are just a cup-shaped filter membrane resting on a below the pore and stay there when the centrifuge stops. The filter plate has a center filter plate supported by a specially designed and six small holes arranged hexagonally. polycarbonate insert. The insert provides space for the filtrate while ensuring that The rounded end of the insert has a radius of 6.18 mm, which is slightly larger than none of it can get back to the membrane when the centrifuge is stopped. When an that of the inside of the centrifuge tube (6.08 mm). This ensures compression stress insert is made a close fit for the centrifuge tube, fluid gets between the insert and the as in the case of the filter for the 25.1 rotor. To set up the litter, the membrane-centertube and can rise to the membrane by capillarity. To prevent this, the insert was pro- ing collar is fitted to the centrifuge tube and the membrane placed on it. After adding vided with a l-mm annulus on its outside. During centrifugation fluid enters both the the plunger guide, the plunger is used to press central cavity and the annulus, and tends to the membrane into place. Then the guide and collar are moved up the plunger, and the fill the annulus while leaving the central
271
ULTRACENTRIFUGALRECEFTORFILTERS
top part of the centrifuge tube around the membrane cup is rolled between the finger and thumb with considerable pressure to set the folds in the membrane. The plunger is removed, and the filter is ready to accept 0.5 ml of fluid. At 50,000 r-pm, the centrifugal force is 244,000g at the membrane. The plunger and plunger guide were made of Plexiglas, the membrane centering collar of Teflon, and the plate and insert of polycarbonate. Testing Procedures The test material used was a suspension of nicotinic acetylcholine receptor-bearing membrane fragments prepared as before (45). Protein was estimated spectroscopically (6). In determining the effect of centrifugal force on fluid retention, the filters were accelerated to the desired speed and held there for 1 min and the centrifuge was allowed to decelerate as usual. The holdup was determined by subtracting the mean weight of three air-dried filters using 10 mM 4morpholinepropanesulfonic acid buffer, pH 7.4, alone from the mean weight of three similar filters in which the buffer also contained 0.5 M KBr. RESULTS
The l-in. Filter in the SW 25.1 Rotor Using the original membrane support ring and a loading of 0.75 ml on each filter, liquid holdup was not significantly affected by prolonging the time at 25,000 rpm. The values were 1.36, 1.53, 1.58,and 1.60plat0,5, 15, and 30 min, respectively. The results for four different determinations of liquid holdup as a function of speed are shown in Fig. 3A. For the bottom curve, a filter loading of 0.5 ml was used while 0.75 ml was used for the other three. In all four experiments a mean standard deviation for each whole experiment was determined. The square points were determined using the original smooth membrane-forming ring. The points show greater scatter (SD
= 2.82%). The circles were determined using the castellated membrane-forming rings giving an overall standard deviation of 1.13%. The difference between the two standard deviations was significant. As mentioned earlier, at the higher speeds the membrane tends to slide down the surface of the membrane-shaping ring to form a circle of folds or creases at the bottom of the ring where it joins the plate. The process can be seen to start at opposite sides of the bottom of the cup where the slots in the filter plate are tangential to the circumference of the bottom of the cup. To try to prevent crease formation, two slots in the filter plate on each side were filled and machined flat. The results of this are shown by the crosses (Fig. 3A). This method tended to reduce crease formation and at the lower speeds made little difference to liquid holdup but at 20,000 and 25,000 rpm it increased liquid holdup. The bottom curve was obtained by loading 0.5 ml instead of 0.75 ml and immediately centrifuging the filters. The holdup was less presumably because the liquid did not have sufficient time to saturate all of the filter. All of the curves relating liquid holdup to speed are concave upward and appear to be approaching an asymptote although they are not simple exponentials. When the log of holdup was plotted against speed there was some residual concavity. Figure 4 shows that with up to 0.4 mg/filter or 0.53 mg/ml of original solution, the holdup is a linear function of the tissue load. Extrapolation of the least-squares regression line gives the holdup by the unloaded membrane as 0.96 ~1. An attempt to measure this directly by filtering 0.5 M KBr alone gave a value of 0.64 ~1, and the calculated volume of the pores in the membrane (see Discussion) was 0.46 ~1. The OS-in. Filter in the SW 50.1 Rotor The crosses (Fig. 3B) indicate ship between fluid holdup and the 0.5-in. filter in the 50.1 head. were obtained by using the filter
the relationspeed using The crosses as described
272
FREUNDLICH
AND TAYLOR
B
I 5
I 10
I 15
I 20 SPEED
I 25 IN THOUSANDS
I
I
I
I
I
10
20 OF RPM.
30
40
50
FIG. 3. The relationship between the amount of liquid retained on the filter membrane and the t-pm. (A) Using the SW 25.1 rotor (Fig. 1). An aliquot of 0.75 ml was placed on each filter except in the case of the lowest curve where it was 0.5 ml: 0, using the smooth membrane-forming ring; 0, using castellated ring; +, using castellated membrane-forming ring and partly filled-in support disk; A, using 0.5 ml and smooth membrane-forming ring. (B) +, using 0.5 ml and the SW 50.1 rotor; 0, same with a disk of polypropylene gauze between the filter membrane and the filter disk. The last points on each curve were associated with some collapse of three out of six of the centrifuge tubes in each case.
and 0.5 ml of fluid. The circles were obtained by the same technique except that there was a disk of polypropylene gauze between the filter membrane and the filter disk in each case in order to see if the liquid holdup could be decreased by reducing the area of contact between the filter membrane and its support. In fact, this had very little effect. The line in Fig. 3B has been drawn through the means of the first four points. The last two points were associated with partial collapse of half of the tubes in both cases. The collapse was associated with irregularities in centrifuge tube wall thickness in all cases. Up to 25,000 r-pm, the circles (Fig. 3A) using the l-in. filter fit the curve of Fig. 3B closely. Liquid Holdup Using the Suction Filtration Method
Using the 0.5 filtration method
M
KBr method the suction (4) (in triplicate) gave a
value of 2.96 ~1 using 0.75 ml of fluid and 2.45 ~1 using 0.5 ml of fluid. Soluble Protein in the Filtrates
Since it was possible that 0.5 M KBr might solubilize some of the insoluble protein, filtrates from 12 pooled filtrates from controls containing 0.5 M KBr were compared to similar pooled filtrates from controls containing no KBr. The KBr solution contained 0.24 mg/ml and that from the controls the same amount of soluble protein. DISCUSSION
When the buckets are opened after a run at 25,000 rpm, the membranes can be seen to be moist with more moisture present at the lower part of the membrane and especially where the membrane has been in contact with the grid of the filtration plate. The upper parts of the membrane are usually
ULTRACENTRIFUGAL
--A -6
I
)0
I
/
0.2
0.1 DRY
TISSUE
WEIGHT
I
0.3 PER
FILTER,
0.4 mq
FIG. 4. Relationship between the amount of liquid retained and the amount of particulate matter placed on the filter using the SW 25.1 rotor. The least-squares regression line cuts the vertical axis at 0.96 ~1. Point A represents the liquid retained when 0.75 ml of 0.5 M KBr was used containing no paniculates. Point B represents the calculated volume of liquid required to fill all the membrane pores.
relatively dry. It appears that there is residual fluid at least on some of the membrane surfaces, in some of its pores, and where the membrane is in contact with the supporting material of the filter itself. The 0.2-pm pore density of the lo-pmthick membranes is given as 3 x 10s crne2 (7) corresponding to a total pore volume of 0.46 @membrane. This accounts for about half of the experimentally determined holdup of 0.96 ~1 obtained by extrapolation of the data in Fig. 4. The somewhat lower value of 0.6 ~1 obtained by filtering 0.75 ml of 0.5 M KBr in 10 mM 4-morpholinepro-
RECEPTOR
FILTERS
273
panesulfonic acid buffer may have been due to incomplete wetting of the filter membrane. All the curves of holdup versus speed seem to be approaching an asymptote and this was particularly true in the case of the curve (crosses) in Fig. 3A where the area of contact between the filter membrane and the support disk had been increased. Some of the irreducible holdup must be due to surface imperfections in the machined surfaces of the filter. The situation might be improved by polishing the upper surface of the membrane-forming ring of the l-in. filter or the plate of the OS-in. filter. Thinner membranes with smaller pores are available, but have not been tried. Higher tissue concentrations in smaller volumes would improve the signal to noise ratio. A centrifuge rotor which goes up to 65,000 rpm with buckets taking 0.5-in. tubes is available. Treating all but a circle of 1 cm diameter of the 25-mm filter membranes with a plastic spray to fill the holes would allow adequate filtration, but would reduce holdup. REFERENCES 1. Bennett, J. P., Jr. (1978) in Neurotransmitter Receptor Binding (Yamamura, H. I., Enna, S. J., and Kuhar, M. J., eds.), pp. 57-90, Raven Press, New York. 2. Taylor, D. B. (1973)J. Pharmacol. Exp. Ther. 186, 537-551. 3. Helfferich, F. (1962) Ion Exchange, McGraw-Hill, New York. 4. Spivak, C. E., and Taylor, D. B. (1977) Anal. Biochem. 77, 274-279. 5. Franklin, F. I., and Potter, L. T. (1972) FEBS Letr. 28, 101. 6. Kalb, V. F., and Bernluhr, R. W. (1977) Anal. Biochem. 82,362-371. 7. General Electric Catalogue (1978) Lab 40.