U.S. patent number 3,620,947 [Application Number 04/815,145] was granted by the patent office on 1971-11-16 for electrophoretic system.
This patent grant is currently assigned to Ortec Incorporated, Oak Ridge, TN. Invention is credited to Dorothy J. Moore, Robert Carter Allen.
United States Patent |
3,620,947 |
|
November 16, 1971 |
**Please see images for:
( Certificate of Correction ) ** |
ELECTROPHORETIC SYSTEM
Abstract
An electrophoretic system for concentrating and separating
charged particles and for improving the resolution of the separated
particle laminae that utilizes a buffer composition which will
provide a ratio of leading-trailing ion boundary to the fastest
particle mobility that approaches unity, during the period of free
electrophoresis.
Inventors: |
Robert Carter Allen (Oak Ridge,
TN), Dorothy J. Moore (Oak Ridge, TN) |
Assignee: |
Ortec Incorporated, Oak Ridge,
TN (N/A)
|
Family
ID: |
25216995 |
Appl.
No.: |
04/815,145 |
Filed: |
April 10, 1969 |
Current U.S.
Class: |
204/467;
204/468 |
Current CPC
Class: |
G01N
27/44756 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); B01k 005/00 () |
Field of
Search: |
;204/180R,180G,299 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Schwalbe, "Pulsed Field Electrophoresis," Medical Electronics,
Internat. .
Conf. on Med. Electronics, Paris, 1959, QT34 I64m (1959) .
Davis, "Method and Appt. to Human Serum Proteins," Annals of the
N.Y. .
Academy of Sciences, Vol. 121, Article 2, Page 424 Dec. 28,
1964..
|
Primary Examiner: John H. Mack
Assistant Examiner: A. C. Prescott
Attorney, Agent or Firm: Ralph L. Cadwallader Lawrence P.
Benjamin
Claims
1. In an electrophoretic process for concentrating and then
separating certain components of a mixture of like sign-charged
particles through a sieving gel medium, the gel medium having a
plurality of contiguous zones; the particles suspended in solution
adjacent the gel medium; having a pair of electrodes of opposite
polarity, each electrode positioned at opposite ends of the gel
medium; a source of power for connection to the electrodes; and a
buffer solution surrounding the gel medium and the electrodes, the
improvement comprising: initially positioning the particle solution
between two gel zones; providing a source of ions of like sign and
of different mobilities in both the buffer solution and the gel
medium; providing the faster ion in the gel medium and the slower
ion in the buffer solution; and applying a source of constant,
pulsed DC field across the gel medium during
2. The process of claim 1 further comprising: providing a single
gel zone between the particle solution and the buffer solution;
lowering the conductance of both the single gel zone and particle
solution to raise the voltage gradient appearing thereacross; and
maintaining all gel zones, the particle solution and buffer
solution at the
3. The process of claim 2 wherein the buffer solution is a member
of the group consisting of citrate-borate, sulfate-borate and
chloride-borate.
4. The process of claim 3 wherein the buffer solution is
citrate-borate.
5. The process of claim 3 wherein the buffer solution is
sulfate-borate.
6. The process of claim 3 wherein the buffer solution is
chloride-borate.
Description
This invention relates to an electrophoretic system and more
particularly to an improved electrophoretic system for producing
greater resolution of the separated particle laminae.
Electrophoresis, particularly that carried out in a molecularly
sieving medium such as starch gel and more importantly in
acrylamide gel, represents one of the most effective procedures for
the separation of charged particles. However, a major problem in
such gel electrophoresis, whether carried out with either single
ion buffer systems or discontinuous buffer ion systems or in
discontinuous pH and buffer ion combination systems, has been in
the separation and consequent quantification of isoenzymes, i.e.,
multiple molecular forms of the same enzyme. (Early qualitative or
quantitative changes in a particular isoenzyme profile of an
individual may have great significance in the early detection of
physiological changes associated with a disease such as cancer,
etc., prior to the onset of the present commonly recognized
clinical signs.)
The drawback of a continuous buffer system resides in the fact that
one must use low current densities and voltage gradients to limit
joule heating to levels below that which may destroy enzyme
activity. This serves to lengthen separation time with a resultant
loss in zone resolution. In addition, excessively long (10 cm.)
separations must be performed to obtain adequate resolution.
The use of a discontinuous pH and buffer ion systems, wherein
samples to be separated are first polymerized in an anticonvection
large pore gel medium, may lead to enzyme inactivation due to free
radical formation in the polymerization procedure. In this
situation, up to 65 percent of some types of enzyme activity may be
lost in such a procedure. While current densities utilized in
protein separations with this technique may be employed at levels
sufficient to perform separations up to 21/2 cm. in length in 30 to
40 minutes, current and voltage must, nevertheless be markedly
reduced for enzyme separations of even the more heat stable
isoenzymes such as esterases. Thus, separations of 75 to 90 minutes
are required for acceptable resolution.
With discontinuous ion and pH systems, which require trailing ion
migration to be programmed by means of pH differences, and where
the counter ion is Tris (hydroxy methyl amino methane), lowering
the temperature of the system to separate heat labile enzymes
increases the pH of the system approximately 0.03 pH units per
degree Centigrade of temperature decrease. Thus, a buffer made up
at the proper pH at room temperature will be about 0.7 pH unit
higher at 1.degree. C. Since the resolution in the discontinuous pH
and ion system depends in major part on the trailing ion mobility
being programmed by appropriate pH to be less than that of the
slowest particle to be separated during the stacking phase,
lowering the temperature sufficiently to protect many enzymes will
raise the pH and thus cause the trailing ion to migrate faster than
the slower particles, thereby degrading separation quality. The
necessity to carefully adjust buffer ion concentrations for the
different temperature conditions utilized with this technique for
protein and isoenzyme separations severely limits the versatility
of this systems.
When it is necessary to separate isoenzymes and proteins of both
plasma and tissues, under identical conditions for accurate
qualitative and quantitative comparison, a multiple sample gel slab
rather than individual separations is most desirable and may, in
certain instances, be required.
A thin gel slab is desirable also for the following additional
reasons: (1) temperature and voltage are identical across the
entire slab, and (2) slabs, due to their flat surfaces, are
considerably more suitable for densitometric analysis due to the
absence of combinations of vertical alignment usually encountered
with cylindrical shapes.
The present invention provides an electrophoretic system for
concentrating and separating charged particles resulting in an
improved resolution of the separated particle laminae by utilizing
a buffer combination which provides a ratio of leading-trailing ion
boundaries to fastest particle mobility that approaches unity,
during the period of free electrophoresis. In our system, the
buffer combination and pH range, together with the gel buffering
concentration is carefully chosen so that a discontinuous buffer
ion and continuous gel pH system is provided to produce the
enhanced resolution of separated particle laminae.
It is, therefore, one object of the present invention to provide a
two-step, continuous pH, discontinuous buffer ion electrophoresis
system, wherein charged components are separated from a starting
medium to produce thin laminae in a similar pore separating gel by
utilizing a conductivity shift.
Another object of the present invention is to provide a two-step,
continuous pH, discontinuous buffer ion electrophoresis system
wherein two sequential, rapidly moving ionic boundaries pass
through the components that had been previously separated,
resulting in a sharpened zone as the boundaries formed at the
leading and trailing ions pass through each zone.
Another object of the present invention is to provide a two-step,
continuous pH, discontinuous buffer ion electrophoresis system
wherein an enhanced resolution of various, similar size particles,
in a mixture of many sizes of particles is achieved by providing
gradient pore size gels so that particles differing only slightly
in size with similar charges, may be maximally resolved.
Still another object of the present invention is to provide a
two-step, continuous pH, discontinuous buffer ion electrophoresis
system utilizing a continuous pH gel and a discontinuous buffer ion
solution to enhance the resolution of the separated particle
laminae.
Yet another object of the present invention is to provide a
two-step, continuous pH, discontinuous buffer ion electrophoresis
system capable of producing increased resolution by selection of
leading-trailing buffer ion and gel combinations such that
conductivity differences may be used to effect changes in
separation time and resolution in various areas, as may be desired
by the operator.
A further object of the present invention is to provide a two-step,
continuous pH, discontinuous buffer ion electrophoresis system
which allows simultaneous separation of various types of particles
on the same gel slab under a set of conditions for maximal
resolution for each sample, to allow for the simultaneous analysis
of proteins and enzymes from plasma together with tissue extracts
from various species.
The features of our invention which we believe to be novel are set
forth with particularity in the appended claims. The invention
itself, however, both as to its organization and method of
operation, together with further objects and advantages thereof,
may best be understood by reference to the following description
taken in combination with the accompanying drawings.
FIG. 1a is an exploded view of the buffer tanks and electrodes for
our flatbed, vertical, continuous pH, discontinuous buffer ion
electrophoresis system;
FIG. 1b is the typical cell configuration for use with the tank of
FIG. 1a;
FIG. 2 is a isometric representation of the gel portion of our
electrophoretic system after insertion of a sample but before
application of the appropriate electrical potential;
FIG. 3 is a isometric representation of the gel portion of the same
system after power has been applied for a given duration and shall
hereinafter be referred to as "phase 1";
FIG. 4 is an isometric representation of the system of FIG. 3 after
electrical power has been applied for a longer given period than in
FIG. 3 and is hereinafter referred to as "phase 2"; and
FIG. 5 is an isometric representation of the system of FIGS. 3 and
4 after the separation process has been completed.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring now to FIGS. 1a and 1b, there is shown the tank system 10
for performing our novel electrophoresis process. Lower tank 12 is
provided with sidepieces 12.5, end walls 12.3 and an electrode
12.2. End walls 12.3 are further provided with shoulders 12.4 so
that when the lower tank 12 is filled with an appropriate buffer
solution, (not shown) upper tank 14 will be properly positioned
therein. Electrode 12.2 is preferably made of platinum or some
other inert material that is not affected by either the buffer
solution nor the passage of current therethrough and terminates in
and is connected to a jack 12.1 which passes through one wall 12.3
so that an appropriate source of power may be applied thereto.
Upper tank 14 is also provided with sidewalls 14.6 as well as end
walls 14.2 which end walls are also provided with appropriate
slotted portions 14.3 for accepting and holding the upper electrode
or cathode wire 16.2. The bottom or floor member 14.4 is provided
with a plurality of apertures 14.5 so that cell members 14.51 may
be placed therein. To complete the device, upper electrode 16 is
provided and consists of a platinum wire 16.2 supported by
dielectric member 16.1. The upper electrode or cathode wire 16.2 is
provided with a loop or some other appropriate means for connection
to jack 14.1 so that power may be applied thereto by any of the
many well known conventional methods. One example of such source of
power is disclosed in the copending U.S. Pat. Application Ser. No.
736,642 of Robert H. Dilworth, III, entitled "Process and Apparatus
for Particle Separation," filed on June 13, 1968 and assigned to
the assignee of the subject application.
To operate our device, cell 14.51 is provided with an upper flange
portion 14.52 and a body portion 14.53 having a slotted tappered
portion 14.54 extending the length thereof.
A sample, as will hereinafter be described with regard to FIGS. 2-5
is prepared and placed in the slotted portion 14.54 of cell 14.51.
Thereafter, the underside of flange portion 14.52 is provided with
a nonconductive fluid seal and cell 14.51 is inserted into aperture
14.5 of upper tank 14. It should be apparent, to those skilled in
the art, that the unused sample cell apertures in the bottom wall
14.4 of tank 14 are blocked or otherwise covered if not in use.
Thereafter, lower tank 12 is filled with the selected buffer
solution, to the desired level, and upper tank 14, with the
inserted cells 14.51, is placed into tank 12 to rest on shoulders
12.4. Upper tank 14 is then provided with a sufficient amount of
buffer solution, electrode support 16 is placed in notches 14.3 and
the looped portion of cathode 16.2 is connected to jack 14.1. The
system is now ready for operation upon the application of a
suitable potential across jacks 12.1 and 14.1.
Referring now to FIGS. 2-5, here is shown an isometric
representation of a rectangular cell as it would be when completely
immersed in cold buffer in the lower tank 12 (FIG. 1) and
containing a typical vertical gel slab 18 capable of separating
from one to twelve samples FIG. 2 denotes the typical
electrochemical setup of the system after appropriate amounts of
the sample 20 have been placed in well portion 22 and prior to the
application of electrical energy. FIG. 3 illustrates the initial
separation and concentration, beginning with the application of
power and due to the conductivity shift phase of the separation.
The particles have all been initially partially concentrated and
separated prior to the trailing ion boundary 24 and leading ion
boundary 26 overtaking the particles 28, even though the ionic
boundaries are moving (during the conductivity shift phase) at five
to 10 times the velocity of the particles to be separated. At this
point, the power is gradually increased by increasing the pulse
rate at which the power is applied.
Following this step in our procedure the moving boundary formed by
the trailing and leading anion boundary layers 24 and 26 passes
sequentially through each lamina of protein or particles 28
separated and molecularly sieved in phase 1 of our procedure.
Each lamina 28 is sequentially caught in a zone 32, 34 or 36 that
has been depleted of trailing and leading ions and thus having a
high-voltage gradient. As this high-voltage gradient region moves
through the lamina, the rear portion of the lamina, which the
high-voltage gradient region encounters first, is speeded up and
moves faster than the front portion of the lamina which has not yet
been reached by the high-voltage gradient region. This process
effectively compresses each successive lamina into an ultrathin
layer as the high-voltage gradient region of the moving boundary
passes through.
After the boundary produced by the trailing and leading ion 24 and
26 has passed through all the protein zones 30, 32, 34 and 36, the
ultrathin protein laminae 28 travel at their own mobilities as in
conventional acrylamide electrophoresis.
FIGS. 4 and 5 show the leading 26 and trailing 24 ion boundaries
after passing sequentially through a portion of the laminae
initially separated in FIG. 3, where a resharpening of laminae has
taken place along with a greatly increased lamina mobility. At this
time a maximum pulse rate of constant power consistent with maximal
resolution is employed and FIG. 5 illustrates a completed plasma
protein separation.
The system illustrated takes particular advantage of both the
conductivity shift procedure and the moving ion boundary, along
with the molecular sieving effects of the gradient gel, as shown in
this illustration, for maximum resolution in regions of both high
and low molecular size particles. Thus, band widths as small as 25
to 50 microns by actual measurement are found to be present
following separation. With this technique, 25 or more protein bands
can be resolved from human plasma over a distance of 3.5 to 4
centimeters in 40 to 60 minutes under conditions sufficiently mild
so that easily inactivated substances such as mouse plasma
esterases and tissue extract isoenzymes suitable for quantitative
studies can be separated simultaneously on the same gel.
In comparing the electrochemistry of this disclosure with the
method described by Ornstein and Davis in U.S. Pat. No. 3,384,564,
particular attention must be paid to their FIGS. 1, 2, and 3 and
our FIGS. 2-5. It is evident that, at all times, as shown in their
FIGS. 1 and 2 that the totality of the protein sample or particles
to be separated, is located or sandwiched between the trailing
glycine ion and the leading chloride ion, in a zone relatively
depleted of leading and trailing ions; thus all particles or
proteins are in a region of high-voltage gradient. However, this is
in a supporting medium with a pore size that is large in relation
to the diameters of the various proteins so the proteins will
separate in this zone on the basis of their charge density alone
with no separation based on size. Once this whole region, i.e., the
sandwich of glycine, protein and chloride, reaches the separating
gel region of pH 8.9 the proteins are slowed down by the sieving
action of the small pore size gel and the glycine mobility
increases due to the increase in pH. The glycine then passes
through the entire stack of proteins to migrate directly behind the
chloride ion. From this point on, separation is carried out by
conventional electrophoresis. However, an ultrathin starting zone
consisting of all the proteins or particles was produced at that
point in time when the glycine started to pass through the
proteins.
Also it should be noted in their procedure (FIG. 1) that prior to
the application of electrical energy to the system the trailing
glycine ion (located in the upper buffer reservoir) is in direct
physical contact with a part of the protein or particles to be
separated. Thus the "steady state stacking," the basis of the thin
starting zones and resolving power of their technique, takes place
in a sandwich bounded by the leading and trailing ions between the
two ion boundaries.
BUFFER SELECTION
It has been determined that the resolution of these techniques
depends on the selection of the discontinuous buffer ions. As
indicated in table 1, the mobility of the leading and trailing ion
boundaries at a given power level is dependent on the combined
transport numbers of the ions involved, rather than on the free
mobility of the leading ion alone. Thus, under identical power
conditions the chloride-glycine front mobility is greater than
sulfate-glycine, which in turn, is greater than citrate-glycine,
while chloride-borate is greater than sulfate-borate which is
greater than citrate-borate, even through the mobilities of the
leading ions alone are in the reverse order of mobility. As
indicated in table 1, the greater the mobility of the ion fronts,
the greater is the ratio of front mobility to the mobility of the
particles to be separated. From the two extreme cases stated, i.e.,
chloride-glycine where the fronts are farthest separated and
citrate-borate where the fastest particle (a prealbumin) travels
directly on the trailing ion front, overall resolution was found to
be best when the ratio of the ion fronts' mobility to the fastest
particles's mobility was closest to one. The most striking example
appears in some esterase isoenzyme separations of tissue extracts.
The choice of the best leading and trailing ions for maximal
resolution in our system can be determined to be related to the
ratio of the mobility of the ion boundary to that of the fastest
particle to be separated after such particle has migrated a unit
distance. Thus the lowest ratio tends to produce the best
resolution. ##SPC1##
A study of the conductance of these various systems suggests only
slight differences, if any, in electrochemical behavior between
systems utilizing a conductivity shift and discontinuous buffer
ions at a continuous pH for at least 20-30 minutes. However, in the
case of chloride-glycine, sulfate-glycine and sulfate-borate, the
distance between the ion fronts increases with time. There is then
a decrease in conductance, i.e., an increase in cell impedance.
This is most marked in the chloride-glycine system where the ion
fronts are separated by about 3 millimeters by the time the albumin
front has migrated about 3.5 millimeters with a resultant drop in
conductance of approximately 33 percent. On the other hand, the
sulfate-borate fronts separated by only 0.25 to 0.35 millimeters
during the same time the albumin migrates a similar distance, with
a drop in conductance of less than 5 percent. Thus, the
sulfate-borate system appears to be more desirable as it produces a
higher field strength across the boundary, throughout the
separation process. There is, however, a marked difference between
those systems utilizing discontinuities in both the buffer ions and
pH and in those not utilizing a conductivity shift.
The effect of the conductivity shift in the initial phase of
separation indicates that this is a necessary part of the total
electrochemistry of the system for optimal resolution of various
particle laminae, particularly in the high molecular weight
macroglobulin and haptoglobin regions and in the lower molecular
weight alpha-1-globulin region.
Thus, for optimal resolution in a minimal running time, either the
sulfate-borate or citrate-borate leading-trailing ion combination
is preferred, with the sulfate-borate system being somewhat faster.
Furthermore, borate, sulfate and citrate ions do not produce an
initial pink background in the gel following reactions for esterase
isoenzymes as does glycine. Thus, borate-sulfate or borate-citrate
are also preferable not only in resolution but because they produce
less background for subsequent densitometric studies. However,
citrate, like Tris, has a pH dependency on temperature which makes
sulfate a somewhat better choice for some applications.
SLAB PREPARATION
The polyacrylamide slab (FIGS. 2-5) in cell 14.51 (FIG. 1a) is
prepared in two or more layers of recrystallized acrylamide at the
desired gel concentration and at the same pH, with five layers
typically being used in plasma protein and isoenzyme separations.
As shown in FIGS. 2-5, for plasma separations a lower separating
layer 36 of between 8 -- 8 1/2 percent gel is followed by a layer
34 of between 6 -- 6 1/2 percent gel which is overlayered directly
on layer 36. Layer 34 is then waterlayered to produce an optically
flat surface and a 4 1/2 percent gel layer 32 is cast on top,
waterlayered and allowed to polymerize. Wells 22 are then cast on
top of this layer, with the aid of a well former (not shown), of
between 8 -- 8 1/2 percent gel, at an ionic strength 1/5 to 1/10 of
the previously cast gels. Samples 20 in 50 percent sucrose buffered
to the same pH and ionic strength as the gel columns separating
each well are then placed in the wells. Following application of
the sample, each well is then filled to the top level of the cell
with between 8 -- 8 1/2 percent acrylamide gel, at the lower ionic
strength and consists of the gel cap 30. The interface between the
4 1/2 percent gel and the 6 -- 6 1/2 percent gel should be an
optically flat surface for optimal resolution.
Since acrylamide monomer in the standard commercially available
form contains numerous impurities injurious to several enzyme
systems, optimal separations and reproducibility require the use of
recrystallized monomer.
RECRYSTALLIZATION PROCEDURE
Dissolve 90 grams of acrylamide monomer in a mixture of 300 ml.
ethyl acetate and 300 ml. of benzene and bring to 70.degree. C. in
a water bath.
Filter hot into a heated 1,000 ml. florence flask through Whatman
No. 1 paper in a jacket heated funnel to prevent
crystallization.
Following filtration, the monomer should be recrystallized in an
ice water bath. The temperature of solution in the flask should not
be allowed to drop below 20.degree. C. The crystals are then
removed by vacuum filtration in a Buchner funnel with No. 1 Whatman
paper. The crystals are dried until there is no further odor of
benzene. They are then redissolved in 540 ml. of chloroform and
heated to 60.degree. C. in a water bath. The solution is again
filtered hot and then cooled to no lower than 22.degree. C. and the
crystals removed again by vacuum filtration.
Dropping the temperature in the second (chloroform) step below
22.degree. C. has proven to produce an unsuitable monomer for use
in esterase isoenzyme studies.
Crystals may be stored in the cold by placing them in a dark bottle
and adding just enough chloroform to cover the crystals. Monomer
stored using this procedure has been found to produce suitable
isoenzyme separations after 5 months storage at 8.degree. C. Since
acrylamide is a very reactive monomer, several useful hints and
observations may be used to indicate the shelf life of each
recrystallized batch.
A pH change of 0.4 units from the 4.9- 5.2 pH of the freshly
recrystallized monomer and problems of removal of gel from the
cells are both early signs which indicate that the monomer is
deteriorating and which can be determined before changes in
isoenzyme resolution become apparent.
To cast gels utilizing any of the systems of TABLE 1, the following
gel buffer stock solutions are prepared: ##SPC2## ##SPC3##
The following buffers are applicable: ##SPC4##
Stock solution 6a is a stock persulfate catalyst solution prepared
fresh daily and consists of 0.105 g. ammonium persulfate diluted to
100 ml. Stock 6b is a similar stock persulfate catalyst at double
the concentration of 6a, i.e., 0.21 g./100 ml. Stock solution 7 32
percent acrylamide (if final lower separating layer of 8 percent
gel is desired) 4.8 g. of twice recrystallized monomer 0.12 g.
methylene bis acrylamide H.sub.2 o to a final volume of 15 ml.
This stock solution is prepared fresh daily.
The cell is clamped in an appropriate gelling stand and the stand
leveled. A gasket is usually provided at the base of the cell to
prevent leakage during addition of reagents.
In the upright position approximately 20 ml. of 8 percent gel
consisting of 5 ml. of stock gel buffers 1a, 2a or 3a, plus 5.0 ml.
of stock solution 7, plus 10 ml. of stock solution 6a is mixed in a
beaker at room temperature and poured into the gel. Immediately 3.4
ml. of 6 percent monomer concentration consisting of 1.2 ml. stock
solution 1a, 2a or 3a, 0.90 ml. of stock solution 7, 0.30 ml.
H.sub.2 O and 2.4 ml. of stock solution 6a is carefully overlayered
on top of the first layer. This layer is then carefully
waterlayered from one or both edges of the cell and the two layers
of monomer allowed to polymerize. Two milliliters of a 4 1/2
percent gel consisting of 0.80 ml. of stock solution 1a, 2a or 3a,
0.45 ml. of stock solution 7, 0.35 ml. of H.sub.2 O and 1.6 ml. of
stock solution 6b is added and waterlayered. Following
polymerization, 3.6 ml. of 0.075 molar 8 percent gel consisting of
0.90 ml. of stock solution 7, 0.90 ml. of stock solution 1b, 2b or
3b and 1.8 ml. of stock solution 6b, is added, a well former
inserted with the bottom edges of the well former resting on top of
the 4 1/2 percent layer, and the gel allowed to polymerize for at
least 20 minutes. The well former is then removed and the wells
rinsed with distilled H.sub.2 O and then drained carefully by
inverting the cell. The cell is then placed in the upright position
and the sample (200 to 1,000 .mu.g. of protein per well depending
on the test) in 20 .mu.l. of 0.075 molar 50 percent sucrose from
stock solution 1c, 2c or 3c is placed in the well.
Next, 0.075 molar 8 percent sealing gel is added carefully on top
of each sample layer. This sealing gel consists of 1.0 ml. of stock
solution 7, 1.0 ml. of stock solution 1b, 2b or 3b, and 2.0 ml. of
stock solution 6a. This is allowed to gel and the cell is then
ready for electrophoretic separation.
Solutions 1, 2 or 3 must not be intermixed in the same gel, as each
gel slab contains either all Tris citrate (solutions 1a, 1b and 1c)
or all Tris sulfate (solutions 2a, 2b and 2c) or all Tris chloride
(solutions 3a, 3b and 3c).
Should one wish to perform ascending electrophoresis, the cell is
cast in the inverted position and the polarities at jacks 12.1,
14.1 (FIG. 1a) reversed.
Electrophoresis is usually performed in the vertical position,
preferably with the negatively charged electrode 16.2 (FIG. 1) in
the upper tank 14 and the positively charged electrode 12.2 the the
lower tank 12. The lower tank is filled with Tris-borate buffer at
4.degree. C., giving a pH of 9.35 to 9.40, to a level even with the
bottom of the upper buffer tank 14. The cell or cells 14.51
containing the gel slab 18 and sample 20 are inserted through slots
14.5 in the upper tank 14. To prevent leakage between the upper and
lower tank, the cell end plates are sealed to the floor of the
upper tank by pressing the cell and plate into a bead of silicone
grease or a special sealing gasket (not shown). Then sufficient
Tris-borate is poured into the upper tank to cover the platinum
electrode wire 16.2. Thus, the gel serves as a bridge between the
two electrodes and the cell is completely immersed in a large
volume of cold buffer for additional cooling during
electrophoresis.
An electromotive force is then applied across the cell, preferably
in the form of constant power pulses, such as described in the
aforementioned copending patent application of Robert Dillwoth,
III.
The pulse rate is initially set at a level so that a power level at
about 45 milliwatts per mm..sup.2 gel surface is achieved, e.g.,
for a standard cell, 3.5 mm. width, 100 mm. length and 90 mm.
depth, a peak voltage of about 700 v. is discharged through a 1.0
microfarad capacitor at a rate of about 75 pulses per second. The
power level is then raised to 70 milliwatts/mm..sup.2 (125 p.p.s.)
after 5 minutes. Following completion of the initial conductivity
shift step in the separation, 12-15 minutes, the power is raised in
two steps to 85 and 100 milliwatts/mm..sup.2 at 15 and 20 minutes,
respectively (200 and 300 p.p.s., respectively). The second phase
of separation is carried out for both protein and isoenzyme
separations typically at power levels of 100 milliwatts/mm..sup.2
gel surface, although higher power levels with shorter run times
may be used. Voltage level is kept constant throughout a given
run.
For two-cell operation, power setting of about 400 volts and
similar pulse rates are used during the first stage conductivity
shift, then the power level is raised by bringing the pulse rate to
about 225 p.p.s. and 350 p.p.s. at 15 and 20 minutes,
respectively.
Pulsed power electrophoresis is carried on until the albumin front
migrates a distance of 3.5 to 4.0 cm., a matter of 40 to 60 minutes
depending on whether the divalent sulfate or trivalent citrate ions
are used as the leading ion.
Following electrophoresis the upper buffer tank is removed, the
buffer discarded and the cell removed. Excess sealing grease is
removed from the end plate and the gel is ready for removal from
the cell. A 23 gauge 3-inch long cannula, through which water is
flowing under slight pressure, is inserted down both side edges of
the cell and the gel loosened. The cannula is then moved across
both remaining surfaces to loosen the gel glass seal. The gel can
then be teased out of the cell with the cannula or gently pushed
out from bottom to top with a special plate of slightly smaller
dimensions than the inside cell dimension. A rocking motion
imparted to the plate provides the best results. Thus a gel may be
readily removed from a cell in one to two minutes.
Following removal, the gel may be stained in a common protein dye
(such as Amido Black) in 10 percent acetic acid overnight or for 2
hours at 65.degree. C. and then destained by electrophoresis or
repeated washing in 10 percent acetic acid with about five acid
changes in two days.
Alternatively, the gels may be placed in a pH 6.6, 0.04 M
Tris-chloride buffer at 37.degree. C. for 5 minutes and then into a
dye substrate system of fast blue RR.alpha.-napthyl butyrate at pH
6.6 for 15 minutes for the quantitative development of nonspecific
esterase isoenzymes. Some fifty other isoenzyme staining procedures
previously described are amenable to these techniques of
separation.
While the conditions described in detail here are applicable to
plasma and tissue extract proteins and isoenzymes, it will be
obvious to those skilled in the art of electrophoresis that with
the system described herein, ion selection and pH conditions and
gel pore sizes can be varied widely, making the system suitable for
separations of a wide variety of charged particles. Thus, for
example, positive charged particles will behave in a like manner if
appropriate changes are made in buffer and pH, or separations may
be carried out with negative ions at low or lower pH levels than
those specifically described for plasma and certain tissue extracts
above.
While there has been described what is presently considered the
preferred embodiments of the invention, it should now be obvious to
those skilled in the art that various other changes and
modifications may be made therein without departing from the
inventive concept contained herein, and it is, therefore, aimed to
cover all such other changes and modifications that may fall within
the true spirit and scope of the invention.
* * * * *