An electrophoresis system which provides enhanced resolution and ability to identify nucleases through use of a two-dimensional technique involving isoelectric focussing of tube gels followed by the electrophoresis of second-dimension slab gels formed by the use of a holder allowing slab gels to be cast directly on the sides of the tube gels. DNA is employed as the substrate for casting the slab gels. The slab gels are electrophoresed for the second dimension in a chamber wherein the rack containing a stack of slab gels forms the partition between the anolyte and catholyte compartments. After the second-dimension electrophoresis, the slab gels are incubated in incubation buffer and placed in Pyronin Y to stain the unhydrolized DNA. After staining, they are destained in acetic acid. The DNAses are then visible as colorless spots in a reddish-colored gel.

FIELD OF THE INVENTION 
This invention relates to electrophoretic procedures, and more particularly 
to a two-dimensional electrophoretic technique for providing 
two-dimensional enzyme visualization among proteins in a sample. This 
invention is related to one entitled "Multi-Slab Gel Casting and 
Electrophoresis Apparatus" in the names of Brown, Karpetsky and Jewett. 
BACKGROUND OF THE INVENTION 
Generally, electrophoresis is used to separate complex substances into 
their component parts by using procedures based upon the migration of 
electrically charged fractions in a direct current electric field. 
Previously, this has usually been done using a one-dimensional system in a 
support mediun such as polyacrylamide get with the addition of denaturing 
agents, such as SDS or urea, which provides a separation based on mass or 
on a mass-to-charge ratio. 
Typical prior publications describing the one-dimensional technique include 
the following: 
T. P. Karpetsky et al, "Use of Polynucleotide/Polyacrylamide-gel 
Electrophoresis as a Sensitive Technique for the Detection and Comparison 
of Ribonucleases Activities", (1980) Biochem.J.189,277-284; C. W. Wilson, 
"A Rapid Staining Technique for Detection of RNase after Polyacrylamide 
Gel Electrophoresis", (1969), Anal.Biochem., 31,506-511; C. Wilson, 
"Polyacrylamide gel electrophoresis of corn ribonuclease isoenzymes", 
(1971) Plant Physiol., 48, 64-68; L. C. Van Loon, 
"Polynucleotide-acrylamide Gel Electrophoresis of Soluable Nucleases from 
Tobacco Leaves", (1975), FEBS Lett., 51, 266-269; E. J. Zollner et al, 
"Human Serum Deoxyribonuclease Assay in [.sup.3 H] DNA-polyacrylamide Gels 
without Staining Artifacts" (1974), Anal Biochem 58,71-76; J. B. Boyd and 
H. K. Mitchell, "Indentification of deoxyribonuclease in 
Polyacrylamide-gel following their separation by disc electrophoresis", 
(1965) Anal.Biochem, 13, 28-42; 
SUMMARY OF THE INVENTION 
The technique of the present invention addresses the problem of enhancing 
the resolution of the various components in any given sample run in an 
acrylamide gel by using a two-dimensional system. Better resolution is 
obtained since the isoelectric focussing used for the first dimension 
involves the use of carrier ampholytes in the gel which form a pH gradient 
across the gel when a direct current is applied. This procedure separates 
protein components on the basis of their isoelectric point. The 
polynucleotide/polyacrylamide gel used for the second dimension separates 
proteins on the basis of mass and charge. The use of a two-dimensional 
system enhances the sensitivity so that the use of radioactive substrates 
previously required becomes unnecessary to locate the nuclease activity 
among the proteins separated. 
In addition, the use of electrophoretic techniques for both dimensions 
using non-denaturing conditions, in contrast to previous methodology, does 
not destroy enzyme activity. Thus, enzyme activity may be observed either 
in the gel or after extraction from the gel following the completion of 
the electrophoresis. 
The sensitivity of the method of the present invention is further increased 
by using a high molecular weight substrate in the gel for the second 
dimension rather than diffusing in a low molecular weight substrate; a 
substrate of too high a molecular weight will not penetrate the gel, at 
the completion of the electrophoresis. This eliminates the possibility 
that some of the sharpness of the resolution of the proteins may be lost 
due to diffusion or to broadening of the bands since this is a 
time-consuming process. 
In addition, using the substrate within the gel allows for the detection of 
nucleases in crude samples such as serum and supernatants from sonicated 
cell preparations. Incubation of the gels in buffers of varying 
composition, pH, and ionic strength, aids in obtaining profiles of 
multiple enzyme activities in these samples, with the results being 
superior to those obtained from classical methods used to quantitate 
enzyme activity. 
The electrophoretic technique described herein provides for enhanced 
resolution and ability to identify nucleases through the use of a 
two-dimensional electrophoretic system involving the use of isoelectric 
focussing of a tube gel, followed by electrophoresis in the second 
dimension, which suitably uses a polyacrylamide slab gel containing 
polynucleotide. The second dimension slab gel is cast directly on the side 
of the tube or cylindrical gel. The gels are then incubated in incubation 
buffer and then placed in a suitable stain, such as Pyronin Y, to stain 
the unhydrolyzed polynucleotide. After staining, they are destained 
overnight such as in 7% acetic acid. The nucleases are then visible as 
colorless spots in a colored gel. 
Accordingly, a main object of the invention is to provide an improved 
electrophoretic technique which overcomes the deficiencies and 
disadvantages of the one-dimensional electrophoretic techniques previously 
employed. 
A further object of the present invention is to provide an improved 
electrophoretic technique which employs isoelectric focussing in one 
direction to separate proteins by isoelectric point, and subsequently uses 
polynucleotide/polyacrylamide gel electrophoresis to separate proteins by 
mass-to-charge ratio and to visualize only nuclease activities among all 
proteins. 
A still further object of the invention is to provide an improved 
electrophoretic technique wherein, because proteins are separated in two 
dimensions, the resolution of nucleases is far superior to that obtained 
using a one-dimensional technique, wherein the sensitivity is such that it 
is not necessary to rely on radioactive substrates to locate enzyme 
activity and wherein the sensitivity is increased by using a high 
molecular weight substrate within the gel rather than diffusing it in as a 
low molecular weight substrate after electrophoresis. 
A still further object of the invention is to provide an electrophoretic 
technique of the type above mentioned, wherein DNA is employed in the 
procedure, and wherein DNAses are visualized as colorless spots in a 
colored gel having two-dimensional coordinates, permitting easy comparison 
with normal reference samples.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT 
Referring to the drawings, FIG. 4 is a flow chart generally illustrating a 
one-dimensional technique wherein a sample of cells is washed, sonicated 
and spun, after which the intracellular contents are mixed with glycerol, 
bromophenol blue dye and a running buffer and electrophoresed for about 90 
minutes at 3.degree. C. using a cylindrical gel column containing DNA. 
During electrophoresis the DNAses are inactive and the DNA remains intact 
in the gel column. The material in the column is then allowed to incubate 
in a suitable buffer to activate the DNAses. The DNA is hydrolyzed by the 
DNAses, but the regions of enzyme activity are not visible. Thereafter, 
the cylindrical gel column is stained for 6 hours at 37.degree. C. by 
Pyronin Y in 7% acetic acid. The column is then destained in 7% acetic 
acid, causing the DNAses to be visualized as spaced colorless bands in a 
column of reddish DNA containing gels. 
Referring to FIG. 5, this flow chart depicts the steps involved in 
obtaining two-dimensional visualization as described in the previously 
summarized procedure. FIGS. 1, 2 and 3 illustrate apparatus used in 
carrying out the two-dimensional electrophoresis method of FIG. 5. 
Referring to FIG. 1, 11 generally designates a casting device used for 
forming the slabs from the slab gel solution after the isoelectric 
cylindrical gels have been processed through the first electrophoresis 
stage of the two-dimensional technique. The casing device 11 comprises a 
generally rectangular Plexiglas main housing 12 having a rectangular 
bottom wall 13, a front wall 14, a rear wall 15, and opposite end walls 
16, 16. A forwardly and upwardly inclined shelf member 17 is rigidly 
secured in the bottom front portion of the housing 12, sloping rearwardly 
and downwardly and forming a right-angled corner with an upwardly and 
rearwardly inclined rigid supporting plate member 18 rigidly secured in 
the housing, thereby defining an inclined seat for a Plexiglas rectangular 
frame member 28, defining a gel holder which is slidably engageable in the 
housing. 
The gel holding frame 28 is shaped to receive a stack of rectangular glass 
gel slab plates 19, being clampingly engaged by a rectangular top clamping 
plate 20 which is pressed downwardly by a plurality of spaced clamping 
screws 21 threadedly engaged through the top wall 22 of the frame 28 (see 
FIG. 3). Each slab plate 19 is formed with a longitudinally aligned pair 
of longitudinally extending cylindrical gel-receiving grooves 23, 23 
located adjacent to the rear edge of the plate 19 and on opposing 
surfaces, and hence being at the lowermost part of the associated slab 
plate 19 when the loaded frame 28 is seated in inclined position in the 
seat defined by right-angled plates 17, 18. Respective flat transverse 
Plexiglass spacer strips 25 are adhesively secured on the opposite end 
portions and middle portion of each glass plate 19. Clamping plate 20 is 
provided with recesses or indentations 24 to receive and interlock with 
the bottom ends of the clamping screws 21. 
Rigidly secured on the top wall member 22 of frame 28 is an upstanding, 
longitudinally extending, flat handle flange 26. A large removable top 
wedge member 27 interfits between the front wall 14 and the loaded plate 
holder 28 to prevent formation of a large block of polyacrylamide along 
one side of the plate holder 28 during polymerization. Wedge member 27 is 
provided with a top gripping handle 29. 
Referring to FIG. 3, 30 generally designates an electrophoresis chamber for 
performing the second-dimensional electrophoresis step on the slab gels 
carried in the gel holder 28 after polymerization of the slab gels on the 
glass plates 19, as will be presently described. The chamber 30 comprises 
a generally rectangular Plexiglas housing 31 having a rectangular bottom 
wall 32, front and rear vertical longitudinal walls 33, 34, and vertical 
opposite end walls 35, 36. Symmetrically secured to the inside surfaces of 
the opposite end walls are respective pairs of spaced vertical blocks 37, 
37, defining therebetween opposite vertical guide channels 38 in which the 
opposite ends of the gel holder 28 are slidably receivable. A pair of 
spaced parallel, continuous 1/8" sponge rubber strip gaskets 39, 39 are 
mounted in support grooves provided therefor in the guide channels 38 and 
the bottom wall 32, said spaced strip gaskets being sealingly engageable 
with the end and bottom surfaces of the gel holder 28. A rectangular top 
cover plate 40 has apertures 41 to receive upstanding studs 42 provided on 
the top ends of the blocks 37. Wind nuts 43 are engageable on the studs 42 
to lock the cover plate 40 onto the top of housing 31. Cover plate 40 is 
provided with a longitudinal slot 44 alongside a longitudinal gripping 
handle 45, said slot 44 receiving and providing clearance for the handle 
26 of gel holder 28. Wings screws 46, 46 are threadedly engaged through 
cover plate 40 at the gripping handle 45, said wind screws being 
engageable with the top wall 22 of the gel holder 28 to exert downward 
clamping force on the gel holder 28 to insure sealing engagement thereof 
with the gasket strips 39, 39. 
Respective anolyte and catholyte spaces are thus defined on opposite sides 
of the gel holder 28 when it is installed in the housing 31 as above 
described. Mounted in these spaces are respective electrophoresis 
electrodes 47, 48 supportingly secured to end wall 35 and leading to 
external connection prongs 49, 50 on end wall 35 for connecting the 
electrodes to a suitable d.c. voltage source. 
Referring to FIG. 5, the two-dimensional technique begins with the sample 
preparation, the isoelectric focussing of the tube gel for the first 
dimension and the casting of the slab gel on the side of the cylindrical 
gel for the second dimension. The procedure concludes with the 
electrophoresis of the second dimension and with the incubation and 
staining for the visualization of the nucleases. 
In detail, in a typical but not limitative procedure: 
Glass tubes (110.times.2 mm i.d.) were soaked for a minimum of 30 minutes 
in Chromerge, rinsed four times in glass distilled water and given a final 
rinse in 95% (v/v) ethanol. The tubes were dried at 120.degree. C. Glass 
plates 19 (250.times.25.times.3 mm) were washed with a mild detergent, 
thoroughly rinsed with water, and dried at room temperature. 
Step 1: Samples were prepared by mixing thawed cells with 10 mM Tris-HCl, 
pH 7.4 (sample buffer) to a concentration of 1.times.10.sup.6 cells/5 
.mu.l. The samples were then sonicated for 20 sec. at 45 watts and 
7.degree. C., using a Sonicator Cell Disruptor (Model W225R, Heat Systems 
Ultrasonics, Plainview, N.Y.). After sonication, the samples were 
centrifuged in an Eppendorf Centrifuge for 5 minutes at 40.degree. C. An 
aliquot of the supernatant (usually 5 .mu.l, corresponding to the 
solubilized intracellular contents of 1.times.10.sup.6 cells) was mixed 
with sample buffer to a final volume of 15 .mu.l. To this mixture was 
added 2 .mu.l of a carrier ampholine mix (1:1.5:7 mixture of pH 5-7, 40% 
w/v, pH 9-11, 20% w/v, pH 3.5-10, 40% w/v), LKB Instruments, Inc. 
Rockville, Md., 3 .mu.l of water, and 20 .mu.l 0.01% w/v bromophenol blue 
in 85% glycerol. 
Step 2: Isoelectric focussing gels were prepared using 16 ml of distilled 
water, 6.5 ml of acrylamide stock (30% w/v acrylamide, 0.8% w/v 
bisacrylamide, Bio-Rad Laboratories, Richmond, Calif.), 1.8 ml of carrier 
ampholine mix, 2.85 ml glycerol, and 10 .mu.l N,N,N',N' 
(tetramethylethylene-diamine, referred to as TEMED, Bio-Rad Laboratories, 
Richmond, Calif.). The gel solution was degassed at 12 Torr for 6 minutes. 
Polymerization was initiated by the addition of 1.5 ml of an ammonium 
persulfate solution (Bio-Rad Laboratories, Richmond, Calif.), 1% w/v in 10 
mM Tris-HCl, pH 7.4. Gel tubes were filled to a height of 97 mm, and the 
gel was covered by an ampholine overlay solution (2% v/v carrier ampholine 
mix). Polymerization was complete within 30 minutes. The gels were used 
within two days. 
Step 3: 20 .mu.l of overlay solution was added to each gel before 
commencing the run. Freshly degassed catholyte (10 mM phosphoric acid) and 
anolyte (20 mM NaOH) were added to the upper and lower electrophoresis 
chambers, respectively. 
Step 4: The gels were then pre-electrophoresed at 4.degree. C. at 200 v. 
for 15 minutes, 300 v. for 30 minutes, and 400 v. for 30 minutes. The 
catholyte was then removed, and 20 .mu.l of sample, followed by 20 .mu.l 
of ampholine overlay solution and freshly degassed catholyte were added. 
Electrophoresis was continued at 400 v. for 17-20 hours at 4.degree. C. 
Step 5: Approximately 15 minutes before the isoelectric focussing was 
complete, the slab gel solution containing DNA was made, as follows: 100 
ml distilled water, 57 ml of acrylamide stock solution, 10.53 ml of DNA 
stock solution (5 mg/ml stored at 4.degree. C., Worthington Biochemical 
Corp., Freehold, N.J.), 52.65 ml buffer A (1.5 Tris, pH 8.9), and 88 .mu.l 
TEMED. After mixing, the solution was degassed under a vacuum. 
The cylindrical gels were placed in the grooves 23 (1 mm wide and 0.5 mm 
deep) of the glass plates 19. The glass plates 19 were placed in the gel 
holder 28, which was tilted and placed in the slab casting apparatus 11 in 
the manner previously described. 
To 200 ml of the slab gel solution was added 13.2 ml of ammonium persulfate 
solution. The gel solution was poured into the casting apparatus 11. Air 
bubbles trapped between the plates 19 were released by tapping the 
apparatus. The wedge member 27 was placed in the apparatus 11 against the 
plate holder 28 in the manner above described to prevent the formation of 
a large block of acrylamide. 
The gel polymerized within one hour. The wedge 27 and the gel holder 28 
were removed from the casting apparatus 11. The excess acrylamide was 
trimmed off with a razor blade. 
The ends and bottom of the gel holder 28 were covered with a thin layer of 
high vacuum grease (Dow Corning Corp., Midland, MI). The holder 28 was 
placed in the electrophoresis apparatus 30 in tight contact with the two 
rubber gasket strips 39, 39. The chambers on opposite sides of the holder 
28 were filled with equal volumes of 50 mM Tris-HCl and 0.38M glycine, pH 
8.6 (running buffer), and 0.01% (v/v) bromophenol blue was added to the 
catholyte. 
Step 6: Electrophoresis was accomplished using 12.5 mA per gel at 4.degree. 
C. until the bromophenol blue traversed the width of the slab gels. After 
removal of the gels, one corner was marked to indicate the position of 
each focussed gel. 
Step 7: The gels were then incubated in either 50 mM citrate-phosphate 
buffer, pH 4.0, or 100 mM Tris-HCl, pH 7.4 containing 2.5 mM MnCl.sub.2 
and 0.1 mM CaCl.sub.2, using a volume of 50 ml/gel and placed in a shaker 
(Model G-25, Gyratory Shaker, New Brunswick Scientific Co., Inc. Edison, 
N.J.) at 37.degree. C. Other buffers may be used to detect other types of 
nucleases. The buffer was changed twice at 15-minute intervals. The gels 
were then allowed to incubate in the buffer overnight. The buffer was 
removed and replaced with Pyronin Y (Bio-Rad Laboratories, Richmond, 
Calif.) 0.1% (w/v) in 7% (v/v) acetic acid in order to stain the 
unhydrolyzed DNA. After shaking for 6 hours at 37.degree. C., the gels 
were destained overnight in a Bio-Rad destainer containing 7% acetic acid. 
The gels were placed in 50 ml conical tubes with holes cut in the sides 
and lids for support during the destaining procedure. 
To visualize the proteins, the gels were shaken in 10% trichloroacetic acid 
(w/v) and 5% (w/v) sulfosalicyclic acid (fixer) for 1 hour. They were 
given 15-minute washes in 5% (w/v) acetic acid and 25% (v/v) methanol 
(destaining solution). The gels were then stained with 0.1% (w/v) 
Coomassie Brilliant Blue R 250 (Bio-Rad Laboratories, Richmond, Calif.) in 
5% (v/v) acetic acid and 25% (v/v) methanol for 5 hours with shaking at 
37.degree. C. They were incubated in destaining solution overnight. 
While specific embodiments of an improved electrophoretic technique for 
providing two-dimensional enzyme visualization among all other proteins in 
a sample and an apparatus for carrying out the techniques have been 
disclosed in the foregoing description, it will be understood that various 
modifications within the scope of the invention may occur to those skilled 
in the art. Equivalent steps and materials will be apparent to those 
skilled in the art. Therefore it is intended that adaptations and 
modifications should and are intended to be comprehended within the 
meaning and range of equivalents of the disclosed embodiments. 
By using various polynucleotides (DNA, RNA, etc.) for substances within the 
slab gel, and by using these gels to detect various types of nucleases 
activities in samples of serum, plasma, intracellular contents or other 
fluids obtained from patients of normal individuals, it is possible to 
determine if any nuclease or a combination of nucleases, serve as a 
biomarker for the presence, extent of response to therapy of disease.