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Semiautomatic fractionation of dilute polyacrylamide gels
ANALYTIOAL
RIWHEMISTRY
29, 498-504 (1969)
Semiautomatic
Fractionation
Polyacrylamide
of Dilute Gels
H. C. BIRNBOIM Biology and Health Physics Division, Atomic Energy of Canada Limited, Chalk River, Ontario, Canao!u Received November 6, 1968
Dilute polyacrylamide gels are a useful medium for the electrophoretic separation of high molecular weight ribonucleic acids (RNA) (l-3) and phage particles (4). Several devices have been described for sectioning cylindrical gels (5-8), but the most satisfactory results for dilute gels have required that the gel be frozen, then sliced on a microtome or similar instrument (1,3,9,10). We describe here a semiautomatic device designed to slice these soft. gels into uniform transverse sections 1 mm thick. The instrument does not require that the gels be frozen, and is simple and rapid in operation. To demonstrate the use of the slicer, [14C]-labeled ribosomal RNA was subjected to electrophoresis in 2.7% polyacrylamide gel; in a period of less than 5 min it was possible to cut the gel into 50 uniform slices and collect them into scintillation vials. The radioactivity profiles are shown, CONSTRUCTION
AND
USE OF GEL SLICER
The gel slicer, shown in Figures 1 and 2, consists of two mechanisms: a motor-driven syringe to drive the gel at a constant rate through a cutting block, and a moving blade to shear off disc-shaped sections of gel. Details of its construction are described in the legend to Figure 1, The gel is held in a vertical position during fractionation so that itsconfiguration is similar to that during electrophoresis. The path that the gel travels is of uniform bore. A design in which the gel was forced through an orifice of one-third the cross-sectional area proved unsatisfactory due to compression of the soft gel. The device was tested by slicing a gel which had been used for the electrophoretic separation of ribosomal RNA. This was done as follows.. The gel was removed from the Plexiglas tube and transferred to a 50% (v/v) solution of Liqui-nox detergent (Alconox, Inc., New York, N. Y.) . A glycerol solution (5076, v/v) works equally well. Thus wetted, it did not stick to surfaces with which it came into contact. As a further pre498
FRACTION
OF
POLYACRYLAMIDE
499
GELS
caution, a thin coat of silicone lubricant (stopcock grease, Dow Corning Corp.) was applied to the cutting block, blade, and cylinder. Using a polyethylene tube as a pipet, the gel was introduced into the cylinder of the slicer through the hole in the cutting block (G) (refer to Figure 1). This required that the air bleed valve (J) be open and blade (B) be retracted. The gel was displaced upward at 1 mm/5 set using an infusion pump and syringe containing 30% (v/v) glycerol. The blade was driven by the cam across the cutting block at 5 set intervals, shearing off sections of gel, which were collected in scintillation vials. The cut surface of the gel was lubricated with 35-50 el of water using a second syringe (shown in Figure 2). Proper action of the blade-lifting mechanism was established by appropriate adjustment of the front and rear trippers (E and R) . Tension applied to the ball (D) by a spring governed the downward pressure of the blade. The blade and cutting block were positioned so that the blade moved far enough past the hole to shear the last shred of gel. Once properly adjusted, the mechanism required little attention. OTHER
MATERIALS
AND METHODS
Preparation of gels: Dilute polyacrylamide gels cross-linked with bisacrylamide were prepared according to Loening (1) , with minor modifications. For 20 ml of gel mixture, the following solutions were added: 30% acrylamide, 1.8% bisacrylamide, 1.8 ml; 10 x gel buffer (0.4 M Tris base, 0.2 M Na acetate, 0.02 M EDTA, 0.5% sodium dodecyl sulfate, pH 7.4), 2 ml; 50% (w/v) sucrose, 4 ml; 10% (v/v) tetramethylethylenediamine (TEMED) acetate (pH 7), 0.2 ml. The volume of the solution was adjusted to 19.8 ml with water, and it was gently mixed. Following addition of 0.2 ml of 10% (w/v) ammonium persulfate, the solution was transferred to vertical Plexiglas tubes (0.625 cm i.d. X 7 cm) and allowed to gel. Electrophoresis buffer was equivalent to l/10 strength of 10 X gel buffer, except that sodium dodecyl sulfate (SDS) was present at 0.1% concentration. Low concentrations of SDS were added to inhibit any residual ribonuclease which might be present. Preparation of sample: Cytoplasmic RNA was prepared from HeLa cells that had been grown in the presence of [14C]-uridine for 20 hr (11) ; 100 ,pg of RNA was precipitated with 2 vol of ethanol, air dried, and dissolved in 50 ~1 of electrophoresis buffer containing 7% sucrose. 6ounting of radioactive
containing
[l”C]-labeled
slices: Slices of polyacrylamide
RNA
gel (2.7%)
were collected in glass scintillation
vials
and digested with 0.2 ml of NCS reagent (Nuclear-Chicago, Des Plaines, Ill.) for 30 min at. room temperature. Follbwing the addition of 5 ml
500
H.
INFUSION
C.
BIRNBOIM
PUMP
FIQ. 1. Construction of gel slicer. The device consists of two main parts: (1) a mechanism for passing the gel through the cutting block (G) at a uniform rate, and (2) a mechanism for shearing off sections of gel. (1) The cutting block (G) is fashioned from Teflon and has a l/4” hole which is continuous with the l/4” i.d. of the Plexiglas cylinder (H),. The block has a protruding lip to facilitate collection of slices. (J) is an air bleed valve attached to the bottom of cylinder (H) and (K) is a one-way stopcock. The stopcock is connected via a short length of plastic tubing to an infusion pump (Harvard Apparatus Co., model 975). (2) The slicing mechanism consists of a 6 rpm motor (M) which turns cam (C). The action of the cam advances slide (S), which is an aluminum bar sliding in a brass block. Slide (S) is returned by a spring. Cam follower (F) is a l/2” o.d. bearing. Blade aupport (A) is hinged to slide (S). Shown in the inset is a spring-loaded ball (D) (3/16” diameter steel) which bears upon the sharp edge of (A) and acts as a center spring. It allows (A) to be set in the “up” or “down” position by the tripper mechanism (E, R, T). The “single” cam has 3 steps for positioning slide (S) and blade (B). The sequence is as follows. The action of the cam moves the
FRACTION
OF
POLYACRYLAMIDE
GELS
501
FIG.2. Photographof gelslicer. of scintillator (160 ml Liquifluor, Pilot Chemicals, to 3.8 liters of toluene), the samples were counted in a Nuclear-Chicago Mark I scintillation counter. Although not completely dissolved by this treatment, the samples were uniformly quenched as judged by external standardization. Gel slices containing [32P]-labeled RNA were counted in 5 ml of dioxane-based scintillator (6 gm Omnifluor, New England Nuclear Corp., 155 gm naphthalene to 1 liter dioxane) without prior digestion. blade from its back position in SLep 1 to the dwell position (Step 2). This advances blade (B) across the emerging column of gel and shears off a disc-shaped slice. The dwell position allows time for the slice to be collected. As the cam advances to step 3, tripper pin (T) encounters front tripper (E), whiah lifts blade support (A) to its “up” position, where it is held by (D). As the cam moves to Step 1, the blade passes back over the gel and tripper pin (T) encounters rear tripper (R), which knocks blade support (A) to its “down” position. ,This completes the cycle in 10 sec. For 5 set collections, a double cam is used; the sequence of movements occurs twice per revolution. Blade (B) is fashioned from a 0.010” thick piece of stainless steel and its cutting edge is beveled slightly. The sketch is drawn to scale, .325” = 1”.
602
H.
C.
BIRNBOIM
For oounting SH samples, a variety (3,8,12, 12).
of procedures have been described
RESULTS
To evaluate the utility of the slicer, two experiments were performed. In the first, [14C]-ribosomal RNA was subjected to electrophoresis on a 2.7% gel, following which the gel was sliced and counted. The results are shown in Figure 3a. It may be seen that the ribosomal RNA species were well separated. The majority of counts were recovered in one or two slices. These patterns compare favorably with those in which gels were frozen and sliced on a microtome (3, 9). In a second experiment, to test the uniformity of slice thickness, [32P] -RNA was added to the gel mixture. After the gel had set, it was fractioned into 1 mm slices as
IO
20 FRACTION
30 NUMBER
40
50
FIG. 3. (a) Resolution of ribosomal RNA species following electrophoresis on .a 2.7% polyacrylamide gel. The gel, electrophoresis buffer, and t”Cl-RNA were prepared as described under “Other Materials and Methods.” The sample was applied to the gel in a volume of 50 ~1, then subjected to electrophoresis for 30 min at room temperature. At an applied voltage of 35V, the current flow was .5 mA per gel. (b) Uniformity of slice thickness. A 2.7% polyacrylamide gel, 5.5 cm long, was uniformly labeled with i?‘Pl as described under “Methods” and fractionated into 51 slices. Each slice was counted for 20 min in a liquid scintillation counter. The heavy line is the arithmetric mean, excluding the last fraction, ‘which is the tinal 2-3 mm of the gel; the dotted lines indicate * 10% deviation ifrom the mean.
FRACTION
OF
POLYACRYLAMIDE
GELS
503
described above. The results are shown in Figure 3b. With the exception of the first few and last slices, the counts fall within +- 10% of the mean. Cylindrical gels of other concentrations have also been sliced in this way. Gels prepared with less than 2.5% acrylamide may be sliced directly, but are most easily handled if prepared with agarose as described by Dingman and Peacock (12). Gels containing 15-2076 acrylamide and 0.1% bisacrylamide have also been successfully fractionated. Thus, gels encompassing the wide range of acrylamide concentration used for protein and RNA separations may be readily fractionated. DISCUSSION
The instrument described in this report has been developed for the fractionation of dilute polyacrylamide gels used for the separation of large RNA molecules. For these and other studies, 1 mm thick slices are adequate to resolve the components being analyzed. The device is fairly simple in construction and use, and slices may be collected at 5 or 10 set intervals, depending upon whether a single or double cam is used. Slice thickness can be varied by changing the speed of the infusion pump. Effective use of the instrument requires that it be well cleaned, and that surfaces in contact with the gel be lubricated. The principle of “floating” the gel upward has avoided the necessity for freezing prior to fractionation. It may be possible to perform photometric scanning in a similar fashion. While this manuscript was in preparation, a gel slicer designed to fractionate gels with acrylamide concentrations from 2.5 to 20% was described (13). Its construction is more complicated than the one described, and it requires that the gel be chilled or frozen during fractionation. SUMMARY
A device for slicing cylindrical polyacrylamide gels has been constructed. For the fractionation of dilute gels it has the following advantages: (1) handling of the gel is simple and it need not be frozen; (2) the gel is in the upright position (i.e., the same position as during electrophoresis) during fractionation, which may minimize distortion; (3) fractionation is rapid and could probably be fully automated if desired. ACKNOWLEDGMENT I am indebted to Mr. L. G. Hunter of the Biology shop for the design of the blade-lifting mechanism
working model of the instrument.
and Health Physica and for construction
machine of a
504
H.
C. BIRNBOIM
REFERENCES 1. LOENINC, 2. PEACOCK, 3. BISHOP, (1967).
5.
8.
9. 10. 11. 12. 13.
Bioche>pl.
J. 102,
A. C., AND DINGMAN, D. H. L., CLAYBROOP,
251 (1967). Biochemistry 6, 1818 (1967). J. R., AXD SPIEGELMAN, S., J. Mol.
C. W.,
Biol.
26, 373
C. A., III, EDGELL, M. H., ANI SINSHEIMER, R. L., J. Mol. Biol. 23, 553 (1967). CHRAMBACH, A., Anal. Biochem. 15, 544 (1966). ARONSON, J. N., AND BORRIS, D. P., And. Biochem. 18, 27 (1967). MAIZEL, J. V., JR., Science 151, 988 (1966). GRAY, R. H., AND STEFFENSEN, D. M., Anal. Biochem. 24,44 (1968). WEINBERG, R. A., LOENING, U., WILLEMS, M., AND PENMAN, S., Proc. N&Z. Acad. Sci. U. 8. !%3, 1088 (1967). GRESSEL, J., AND WOLOWELSKY, J., Anal. Biochem. 22, 352 (1968). WARNER, J. R., NERO, R., BIRNBOIM, H. C., GIRARD, M., AND DARNELL, J. E., J. Mol. Biol. 19, 349 (1966). DINGMAN, C. W., AND PEACOCK, A. C., Biochemistry 7,659 (1968). GROVES, W. E’., DAVIS, F. C., JR., AND SELLS, B. H., Anal. Biochem. 24, 462 (1968).
4. HUTCHISON,
6. 7.
T!. E.,
RIWHEMISTRY
29, 498-504 (1969)
Semiautomatic
Fractionation
Polyacrylamide
of Dilute Gels
H. C. BIRNBOIM Biology and Health Physics Division, Atomic Energy of Canada Limited, Chalk River, Ontario, Canao!u Received November 6, 1968
Dilute polyacrylamide gels are a useful medium for the electrophoretic separation of high molecular weight ribonucleic acids (RNA) (l-3) and phage particles (4). Several devices have been described for sectioning cylindrical gels (5-8), but the most satisfactory results for dilute gels have required that the gel be frozen, then sliced on a microtome or similar instrument (1,3,9,10). We describe here a semiautomatic device designed to slice these soft. gels into uniform transverse sections 1 mm thick. The instrument does not require that the gels be frozen, and is simple and rapid in operation. To demonstrate the use of the slicer, [14C]-labeled ribosomal RNA was subjected to electrophoresis in 2.7% polyacrylamide gel; in a period of less than 5 min it was possible to cut the gel into 50 uniform slices and collect them into scintillation vials. The radioactivity profiles are shown, CONSTRUCTION
AND
USE OF GEL SLICER
The gel slicer, shown in Figures 1 and 2, consists of two mechanisms: a motor-driven syringe to drive the gel at a constant rate through a cutting block, and a moving blade to shear off disc-shaped sections of gel. Details of its construction are described in the legend to Figure 1, The gel is held in a vertical position during fractionation so that itsconfiguration is similar to that during electrophoresis. The path that the gel travels is of uniform bore. A design in which the gel was forced through an orifice of one-third the cross-sectional area proved unsatisfactory due to compression of the soft gel. The device was tested by slicing a gel which had been used for the electrophoretic separation of ribosomal RNA. This was done as follows.. The gel was removed from the Plexiglas tube and transferred to a 50% (v/v) solution of Liqui-nox detergent (Alconox, Inc., New York, N. Y.) . A glycerol solution (5076, v/v) works equally well. Thus wetted, it did not stick to surfaces with which it came into contact. As a further pre498
FRACTION
OF
POLYACRYLAMIDE
499
GELS
caution, a thin coat of silicone lubricant (stopcock grease, Dow Corning Corp.) was applied to the cutting block, blade, and cylinder. Using a polyethylene tube as a pipet, the gel was introduced into the cylinder of the slicer through the hole in the cutting block (G) (refer to Figure 1). This required that the air bleed valve (J) be open and blade (B) be retracted. The gel was displaced upward at 1 mm/5 set using an infusion pump and syringe containing 30% (v/v) glycerol. The blade was driven by the cam across the cutting block at 5 set intervals, shearing off sections of gel, which were collected in scintillation vials. The cut surface of the gel was lubricated with 35-50 el of water using a second syringe (shown in Figure 2). Proper action of the blade-lifting mechanism was established by appropriate adjustment of the front and rear trippers (E and R) . Tension applied to the ball (D) by a spring governed the downward pressure of the blade. The blade and cutting block were positioned so that the blade moved far enough past the hole to shear the last shred of gel. Once properly adjusted, the mechanism required little attention. OTHER
MATERIALS
AND METHODS
Preparation of gels: Dilute polyacrylamide gels cross-linked with bisacrylamide were prepared according to Loening (1) , with minor modifications. For 20 ml of gel mixture, the following solutions were added: 30% acrylamide, 1.8% bisacrylamide, 1.8 ml; 10 x gel buffer (0.4 M Tris base, 0.2 M Na acetate, 0.02 M EDTA, 0.5% sodium dodecyl sulfate, pH 7.4), 2 ml; 50% (w/v) sucrose, 4 ml; 10% (v/v) tetramethylethylenediamine (TEMED) acetate (pH 7), 0.2 ml. The volume of the solution was adjusted to 19.8 ml with water, and it was gently mixed. Following addition of 0.2 ml of 10% (w/v) ammonium persulfate, the solution was transferred to vertical Plexiglas tubes (0.625 cm i.d. X 7 cm) and allowed to gel. Electrophoresis buffer was equivalent to l/10 strength of 10 X gel buffer, except that sodium dodecyl sulfate (SDS) was present at 0.1% concentration. Low concentrations of SDS were added to inhibit any residual ribonuclease which might be present. Preparation of sample: Cytoplasmic RNA was prepared from HeLa cells that had been grown in the presence of [14C]-uridine for 20 hr (11) ; 100 ,pg of RNA was precipitated with 2 vol of ethanol, air dried, and dissolved in 50 ~1 of electrophoresis buffer containing 7% sucrose. 6ounting of radioactive
containing
[l”C]-labeled
slices: Slices of polyacrylamide
RNA
gel (2.7%)
were collected in glass scintillation
vials
and digested with 0.2 ml of NCS reagent (Nuclear-Chicago, Des Plaines, Ill.) for 30 min at. room temperature. Follbwing the addition of 5 ml
500
H.
INFUSION
C.
BIRNBOIM
PUMP
FIQ. 1. Construction of gel slicer. The device consists of two main parts: (1) a mechanism for passing the gel through the cutting block (G) at a uniform rate, and (2) a mechanism for shearing off sections of gel. (1) The cutting block (G) is fashioned from Teflon and has a l/4” hole which is continuous with the l/4” i.d. of the Plexiglas cylinder (H),. The block has a protruding lip to facilitate collection of slices. (J) is an air bleed valve attached to the bottom of cylinder (H) and (K) is a one-way stopcock. The stopcock is connected via a short length of plastic tubing to an infusion pump (Harvard Apparatus Co., model 975). (2) The slicing mechanism consists of a 6 rpm motor (M) which turns cam (C). The action of the cam advances slide (S), which is an aluminum bar sliding in a brass block. Slide (S) is returned by a spring. Cam follower (F) is a l/2” o.d. bearing. Blade aupport (A) is hinged to slide (S). Shown in the inset is a spring-loaded ball (D) (3/16” diameter steel) which bears upon the sharp edge of (A) and acts as a center spring. It allows (A) to be set in the “up” or “down” position by the tripper mechanism (E, R, T). The “single” cam has 3 steps for positioning slide (S) and blade (B). The sequence is as follows. The action of the cam moves the
FRACTION
OF
POLYACRYLAMIDE
GELS
501
FIG.2. Photographof gelslicer. of scintillator (160 ml Liquifluor, Pilot Chemicals, to 3.8 liters of toluene), the samples were counted in a Nuclear-Chicago Mark I scintillation counter. Although not completely dissolved by this treatment, the samples were uniformly quenched as judged by external standardization. Gel slices containing [32P]-labeled RNA were counted in 5 ml of dioxane-based scintillator (6 gm Omnifluor, New England Nuclear Corp., 155 gm naphthalene to 1 liter dioxane) without prior digestion. blade from its back position in SLep 1 to the dwell position (Step 2). This advances blade (B) across the emerging column of gel and shears off a disc-shaped slice. The dwell position allows time for the slice to be collected. As the cam advances to step 3, tripper pin (T) encounters front tripper (E), whiah lifts blade support (A) to its “up” position, where it is held by (D). As the cam moves to Step 1, the blade passes back over the gel and tripper pin (T) encounters rear tripper (R), which knocks blade support (A) to its “down” position. ,This completes the cycle in 10 sec. For 5 set collections, a double cam is used; the sequence of movements occurs twice per revolution. Blade (B) is fashioned from a 0.010” thick piece of stainless steel and its cutting edge is beveled slightly. The sketch is drawn to scale, .325” = 1”.
602
H.
C.
BIRNBOIM
For oounting SH samples, a variety (3,8,12, 12).
of procedures have been described
RESULTS
To evaluate the utility of the slicer, two experiments were performed. In the first, [14C]-ribosomal RNA was subjected to electrophoresis on a 2.7% gel, following which the gel was sliced and counted. The results are shown in Figure 3a. It may be seen that the ribosomal RNA species were well separated. The majority of counts were recovered in one or two slices. These patterns compare favorably with those in which gels were frozen and sliced on a microtome (3, 9). In a second experiment, to test the uniformity of slice thickness, [32P] -RNA was added to the gel mixture. After the gel had set, it was fractioned into 1 mm slices as
IO
20 FRACTION
30 NUMBER
40
50
FIG. 3. (a) Resolution of ribosomal RNA species following electrophoresis on .a 2.7% polyacrylamide gel. The gel, electrophoresis buffer, and t”Cl-RNA were prepared as described under “Other Materials and Methods.” The sample was applied to the gel in a volume of 50 ~1, then subjected to electrophoresis for 30 min at room temperature. At an applied voltage of 35V, the current flow was .5 mA per gel. (b) Uniformity of slice thickness. A 2.7% polyacrylamide gel, 5.5 cm long, was uniformly labeled with i?‘Pl as described under “Methods” and fractionated into 51 slices. Each slice was counted for 20 min in a liquid scintillation counter. The heavy line is the arithmetric mean, excluding the last fraction, ‘which is the tinal 2-3 mm of the gel; the dotted lines indicate * 10% deviation ifrom the mean.
FRACTION
OF
POLYACRYLAMIDE
GELS
503
described above. The results are shown in Figure 3b. With the exception of the first few and last slices, the counts fall within +- 10% of the mean. Cylindrical gels of other concentrations have also been sliced in this way. Gels prepared with less than 2.5% acrylamide may be sliced directly, but are most easily handled if prepared with agarose as described by Dingman and Peacock (12). Gels containing 15-2076 acrylamide and 0.1% bisacrylamide have also been successfully fractionated. Thus, gels encompassing the wide range of acrylamide concentration used for protein and RNA separations may be readily fractionated. DISCUSSION
The instrument described in this report has been developed for the fractionation of dilute polyacrylamide gels used for the separation of large RNA molecules. For these and other studies, 1 mm thick slices are adequate to resolve the components being analyzed. The device is fairly simple in construction and use, and slices may be collected at 5 or 10 set intervals, depending upon whether a single or double cam is used. Slice thickness can be varied by changing the speed of the infusion pump. Effective use of the instrument requires that it be well cleaned, and that surfaces in contact with the gel be lubricated. The principle of “floating” the gel upward has avoided the necessity for freezing prior to fractionation. It may be possible to perform photometric scanning in a similar fashion. While this manuscript was in preparation, a gel slicer designed to fractionate gels with acrylamide concentrations from 2.5 to 20% was described (13). Its construction is more complicated than the one described, and it requires that the gel be chilled or frozen during fractionation. SUMMARY
A device for slicing cylindrical polyacrylamide gels has been constructed. For the fractionation of dilute gels it has the following advantages: (1) handling of the gel is simple and it need not be frozen; (2) the gel is in the upright position (i.e., the same position as during electrophoresis) during fractionation, which may minimize distortion; (3) fractionation is rapid and could probably be fully automated if desired. ACKNOWLEDGMENT I am indebted to Mr. L. G. Hunter of the Biology shop for the design of the blade-lifting mechanism
working model of the instrument.
and Health Physica and for construction
machine of a
504
H.
C. BIRNBOIM
REFERENCES 1. LOENINC, 2. PEACOCK, 3. BISHOP, (1967).
5.
8.
9. 10. 11. 12. 13.
Bioche>pl.
J. 102,
A. C., AND DINGMAN, D. H. L., CLAYBROOP,
251 (1967). Biochemistry 6, 1818 (1967). J. R., AXD SPIEGELMAN, S., J. Mol.
C. W.,
Biol.
26, 373
C. A., III, EDGELL, M. H., ANI SINSHEIMER, R. L., J. Mol. Biol. 23, 553 (1967). CHRAMBACH, A., Anal. Biochem. 15, 544 (1966). ARONSON, J. N., AND BORRIS, D. P., And. Biochem. 18, 27 (1967). MAIZEL, J. V., JR., Science 151, 988 (1966). GRAY, R. H., AND STEFFENSEN, D. M., Anal. Biochem. 24,44 (1968). WEINBERG, R. A., LOENING, U., WILLEMS, M., AND PENMAN, S., Proc. N&Z. Acad. Sci. U. 8. !%3, 1088 (1967). GRESSEL, J., AND WOLOWELSKY, J., Anal. Biochem. 22, 352 (1968). WARNER, J. R., NERO, R., BIRNBOIM, H. C., GIRARD, M., AND DARNELL, J. E., J. Mol. Biol. 19, 349 (1966). DINGMAN, C. W., AND PEACOCK, A. C., Biochemistry 7,659 (1968). GROVES, W. E’., DAVIS, F. C., JR., AND SELLS, B. H., Anal. Biochem. 24, 462 (1968).
4. HUTCHISON,
6. 7.
T!. E.,