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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to macromolecule blotting and more particularly to
an improved method for electroeluting and blotting macromolecules from a
chromatographic gel to an immobilizing matrix.
2. Prior Art
Smithies, Biochem. J. 61: 629-641 (1955), showed that starch gel could
serve as a molecular sieve through which zone electrophoresis of proteins
occurs. Since then, there have been constant innovations in the technique
of gel electrophoresis. The introduction of acrylamide gels, discontinuous
buffer systems, the use of sodium dodecyl sulfate (SDS) to disaggregate
protein complexes to be resolved on gels, and the eventual combined use of
SDS in discontinuous buffer systems for polyacrylamide gel electrophoresis
have been major contributions to the development of one of the most widely
used analytical and preparative tools of modern biology.
The main object of these techniques has been to visually demonstrate the
homogeneity or complexity of a protein preparation by following the
appearance of disappearance of a particular "band" throughout a given
experimental procedure. One-dimensional gels were found to be adequate,
provided only relatively simple protein samples such as viruses,
bacteriophages, erythocyte ghost membranes, etc., were being analyzed.
More complex systems demanded greater resolving power and new
two-dimensional gel systems were developed. Today, even the thousands of
polypeptides which are a part of the more intricate proteinaceous samples
can be efficiently resolved.
The task of unequivocally correlating a "band" or "spot" with a recognized
function has often been difficult, and this is even more so when the
resolution of the proteins depends on their denaturation. Nevertheless,
many approaches have been developed which allow the identification of a
specific enzyme, antigen, glycoprotein or hormone receptor, etc., in a
gel. These techniques rely on the ability to maintain at least one of the
following prerequisites: (1) that the polypeptides retain their activity
throughout electrophoresis; (2) renaturation of a denatured polypeptide;
and (3) covalent crosslinkage of the protein in question to a detectable
ligand prior to electrophoresis. Moreover, the actual processing of the
gels entails multiple manipulations and extensive incubations and washing
procedures. This is very time consuming and quite often prone to handling
accidents such as breakage and tearing of wet gels or cracking during the
drying of the gels.
In attempting to overcome some of the problems encountered in analyzing
gels, a new approach has evolved. A number of reports have been published
demonstrating that the well established approach of "Southern-blotting",
i.e., transferring DNA patterns from agarose gels to nitrocellulose
membrane filters, can be applied to protein patterns in polyacrylamide
gels. Intact protein patterns are eluted from the gels and are immobilized
on a filter substratum. The substratum is, in turn, subjected to the same
type of procedures which have been used on gels for "band" or "spot"
identification. However, by transferring electrophoretograms to
immobilizing matrices one may benefit from the following advantages: (1)
wet filters are pliable and easy to handle; (2) the immobilized proteins
are readily and equally accessible to various ligands (since the
limitations introduced in gels by differential porosity are obviated); (3)
transfer analysis generally calls for small amounts of reagents; (4)
processing times (incubations and washings) are significantly reduced; (5)
multiple replicas of the gels may be made; (6) transferred patterns may be
stored for months prior to their use; (7) protein transfers may undergo
multiple analyses. Moreover, the transferred protein patterns are amenable
to analyses which would be otherwise extremely difficult or impossible to
perform on gels.
The term "blotting" today refers to the process of transferring biological
macromolecules such as nucleic acids and proteins from gels to an
immobilizing matrix. The term is often used in conjunction with the
relevant macromolecule, e.g., protein blotting, DNA blotting and RNA
blotting. The resulting filter containing transferred immobilized
macromolecule is known as a "blot" or "transfer" and can be incubated with
a ligand, a procedure which may be referred to as "overlay". Thus, for
example, immuno-overlay, lectin overlay or calmodulin overlay refers to
the incubation of a blot with an antibody, lectin or calmodulin,
respectively.
In general, protein blotting should be viewed as two sequential events,
namely the elution of the polypeptide from the gel and the adsorption of
the eluted material to an immobilizing matrix.
Three main driving forces have been exploited for macromolecule elution.
One is diffusion. Here, the gel containing the macromolecules to be
transferred is sandwiched between two sheets of immobilizing matrix which
are in turn sandwiched between foam pads and stainless steel screens. This
final assembly is then submerged in two liters of buffer and allowed to
sit for 36-48 hours. The result of this incubation is that two identical
replica blots are obtained. This may or may not be an advantage. This
depends on the quantity of macromolecule present and the sensitivity of
the assay to be used. The efficiency of transfer may reach 75% with half
the quantity available for each matrix. Since diffusion should occur in
all directions loss of resolution might be expected. Because all the
macromolecules in the gel are subject to the same diffusive force there is
a bias in the speed of elution in favor of the lower molecular weight
macromolecules.
This speed bias is a disadvantage especially when the purpose of the
technique is to quantify the amount of each component in a particular
sample or in comparing samples. The speed bias is also a disadvantage when
subdetectable amounts of the higher molecular weight macromolecules are
eluted.
The second means of macromolecule blotting is based on mass flow of buffer
(convection) through the gel. This is the traditional procedure described
by Southern, J. Mol. Biol. 98: 503-517 (1975). The gel is placed in a
reservoir of buffer. A membrane filter is applied to the gel and paper
towels are piled onto the membrane filter. The towels absorb the buffer
from the reservoir through the gel and membrane filter. This movement of
fluid serves as the driving force which elutes the proteins out of the gel
which are then trapped in the membrane filter to create the blot. The
advantages of this technique are that it takes less time (2-12 hrs.) than
diffusion blotting, is more efficient, and is inexpensive since a
reservoir is the only apparatus required. The major disadvantage is that
this method of elution is only practical with agarose gels and is less
suitable for use with polyacrylamide gels. A modification of this approach
has been suggested which allows bidirectional blotting, i.e., blotting
with two membrane filters, one on either side of the gel. The time for
efficient solution has been dramatically reduced by applying a vacuum to
facilitate the process--Peferoen, et.al., FEBS Lett. 145: 369-372 (1982).
The most widely used mode for protein blotting (it is quite often found to
be advantageous in nucleic acid blotting as well) is based on
electroeluting the macromolecule from the gel. The concept of
electroelution of macromolecules for blotting was originally described by
Arnheim and Southern, Cell 11: 363-370 (1977). Subsequently, numerous
apparatus designs have been reported and some are commercially available.
The essence of the technique is as follows. A wet filter material is
placed on a gel making sure that no air bubbles are caught within the
filter or between the filter and the gel. The filter and gel are then
sandwiched between supportive porous pads such as "Scotch Brite" scouring
pads, foam rubber or layers of wet blotting paper. The assembly is then
supported by solid grids (usually nonconductive). It is very important
that the gel and filter are firmly held together. This ensures good
transfer and prevents distortion of the protein bands. The supported
"gel+filter sandwich" is inserted into a tank containing "transfer buffer"
and placed between two electrodes. The electrodes are connected to a power
supply. Typical currents employed are in the range of 250 mA. An
economical, yet efficient, design that seems to work reasonably well is
that described by Bittner, et.al., Anal. Biochem. 102: 459-471 (1980).
The advantage of electroelution of macromolecules is that the time needed
for elution is greatly reduced. Additionally, since the electric field
strength used for elution is readily quantifiable and manipulable, the
technique is conducive to the determination of exact and readily
reproducible optimum transfer conditions.
Several apparatus have been reported which utilized different designs and
construction for the electrodes. The design and construction of the
electrode system is important because of the need as pointed out by
Bittner, et.al., for a uniform electric potential, i.e., a homogeneous
field across the entire surface of a chromatographic gel. A homogeneous
field is needed to ensure that the macromolecules in the different lanes
of the gel are uniformly transferred to the immobilizing matrix. Only then
can lane to lane comparisons be made. The ideal way of designing an
apparatus which would exert a homogeneous field on a chromatographic gel
would be to use two parallel metal electrode plates. Because the metal
electrodes would have little resistance in comparison to the buffer
solution any potential applied to the electrodes would be uniformly
distributed across their entire surface, thus providing a uniform electric
field with which to elute the macromolecules from the slab gel. A platinum
electrode is preferred because platinum is not readily degraded by
electrolysis. The use of platinum foil for electrodes is impractical
because of its high cost. Apparatus employing a stainless steel cathode
plate with a platinum wire anode (Stellwag and Dahlberg, Nucleic Acid
Research 8: 299-317 (1980), McLellan and Ramshaw, Biochemical Genetics 19:
647-654 (1981) and two graphite slabs weighing 3.75 lbs. each as anode and
cathode, Gibson, Anal. Biochem. 118: 1-2 (1981) have been reported.
However, operating units with these electrode designs require high
current, e.g., 1.5A for two hours for the graphite slabs.
Bittner reports an apparatus employing 12 mil platinum wire for electrodes
and cites indirect experimental evidence to conclude a homogeneous field
is produced. The electrode is formed by stringing uninsulated platinum
wire vertically 19 cm, horizontally 5.5 cm, again vertically for 19 cm,
again horizontally for 5.5 cm and finally vertically for 19 cm. The
distance of the two outer vertical portions of the platinum wire from the
plexiglass walls of the apparatus are 2.4 cm. While it is not stated it
appears from a diagram of the apparatus that the vertical portions of the
wire forming the anode and cathode are aligned. The electrode assemblies
are positioned very close (1.5 cm) to the gel. To infer that the electric
field produced by this electrode design is uniform over the entire surface
of the chromatographic gel, Bittner, et.al., compared the separation
patterns of nucleic acids on 0.75% agarose gels with the separation
pattern after transfer to the matrix and observed that the electroelution
had occurred without distortion and with little loss of resolution.
However, since nucleic acid transfers from agarose gels is readily
accomplished by the technique of Southern blotting the validity of this
test to conclude that a uniform electric field is generated by this
electrode design is questionable. Even though the transferred patterns
covered only 30% of the slab surface area they extrapolated their
observation to the entire gel surface and concluded that the electrical
field was uniform over the entire gel surface. Since electrode designs
will produce uniform but different electric fields in different areas of a
gel, the validity of extrapolating the observations from one area of a gel
to the entire gel is questionable. Additionally, the fact that some loss
of resolution does occur upon transfer further weakens inferences of a
unifrom electric field.
The efficiency of the transfer of the individual macromolecular elements
from the gel to the immobilizing matrix seems to depend on the chemical
nature of the element, i.e., whether it is protein or nucleic acid, the
composition of the gel and the molecular weight of the individual
elements. Many researchers have reported that smaller molecular weight
fragments from the electrophoretic separation of protein isolates on
polyacrylamide gels are eluted with greater efficiency than larger
fragments. See for example: Burnette, Anal. Biochem. 112: 195-203 (1981),
Gershoni and Palade, Anal. Biochem. 124: 396-405 (1982), Howe and Hershey,
J. Biol. Chem. 256 12836-12839 (1981), McLellan and Ramshaw, Biochem.
Genet. 19: 647-654 (1981). This effect was documented particularly well by
Howe and Hershey. By changing the immobilizing matrix every hour they were
able to show that in two hours the low-molecular weight polypeptides were
efficiently eluted whereas six hours were necessary to elute sufficient
amounts of high molecular weight polypeptides. To illustrate contradiction
in the state of the art, Bittner claims that proteins with a molecular
weight range of 14,000 to 110,000 were eluted and transferred virtually
quantitatively from a SDS polyacrylamide gel. Towbin, et.al., (Proc. Nat.
Acad. Sci. U.S.A. 76, 4350-4354 (1979) reports quantitative transfers in
urea but not in SDS polyacrylamide gels.
A number of suggestions have been made to overcome or mitigate this
molecular weight bias in a transfer, among them (i) the use of reversible
gel crosslinkers (Tas, et.al., Anal. Biochem. 100: 264-270 (1979) (instead
of bisacrylamide), followed by gel depolymerization prior to transfer
(Bolen, et.al., Appl. Environ. Microbiol. 43: 193-199 (1982), Renart,
et.al., Proc. Nat. Acad. Sci. U.S.A. 76: 3116-3120 (1979)); (ii) limited
protease digestion of high molecular weight proteins during
electrophoretic transfer to convert them to smaller more easily elutable
peptides (Gibson, Anal. Biochem. 118: 1-3 (1981)); (iii) addition of
detergent SDS to "transfer buffer" to facilitate elution of high molecular
weight proteins (Erickson, et.al., J. Immunol. Methods 51: 241-249 (1982).
The effect of acrylamide concentration on protein elution has not been
studied. One would expect that the elution of high molecular weight
peptides would be affected by the porosity of the gel matrix (Gershoni and
Palade, Anal. Biochem. 131: 1-15 (1983).
OBJECT AND SUMMARY OF THE INVENTION
To facilitate the description of the objects and the description of the
invention, the following terms are described with respect to the longest
sides (top to bottom) of the nonconductive box in which the
electroblotting is performed:
(1) the frontal plane is the vertical plane which runs from top to bottom
and left to right of the box;
(2) the saggital plane is the vertical plane which runs from top to bottom
and front to back of the box; and
(3) the transverse plane is the horizontal plane which runs left to right
and front to back of the box.
The samples to be separated by electrophoresis are placed at discreet
positions at one end of the gel. The gel is placed in an electric field so
that the samples migrate from the top to the bottom. Each sample defines a
lane down which its components travel and are separated, after which the
gel is placed in a second electric field, perpendicular to its surface,
for the purpose of blotting..
It is an object of this invention to provide an improved method for
producing uniform electric fields for blotting which is both economic and
efficient i.e., requires minimal currents to electroelute the
macromolecules from the chromatographic gels, e.g., polyacrylamide.
It is another object of this invention to provide a method for producing an
electric field which is uniform at each transverse plane of the blotting
apparatus but which varies in a controlled predetermined way along the
frontal plane.
It is a further object of this invention to shorten the time required for
the transfer of large molecular weight macromolecules relative to smaller
molecular weight macromolecules from an chromatographic gel and to
accomplish this without loss of the smaller molecular weight
macromolecules.
It is a further object of this invention to improve the transfer of
macromolecules from chromatographic gels so that quantitative assays in
contrast to qualitative assays within a lane may be performed.
It is yet a further object of this invention to provide a means by which
the intensity of the applied electric field may be measured and the
gradient of the electric field be measured. It is of special importance in
generating gradients to be able to monitor the applied field intensity.
An even further object of this invention is to provide the ability to
continuously control the applied field intensity at each electrode pair,
thus creating a vast combination of gradients. This object, coupled with
the previous object, provides the user with a level of control and
function previously unavailable.
Another object of this invention is to provide a tool by which the user is
capable of measuring the relative field strength and its distribution
within any apparatus. This provides the user with the ability to evaluate
and monitor the fields generated by various electrode arrays.
These and other objects of the invention are achieved by using an electric
field which is uniform over the entire length of a chromatographic gel or
an electric field which varies in a predetermined and controlled way over
the length of a chromatographic gel. The invention consists of multiple
pairs of aligned electrodes of opposite charge, each pair accurately
spaced from its adjacent pair of aligned electrodes. The end pairs of
aligned electrodes must be accurately spaced from the nonconducting
surfaces of the electroelution chamber.
Each aligned electrode pair is connected through a device capable of
varying the applied electric potential. By varying the electric potential
of each electrode pair the electric field along the length of the
chromatographic gel can be uniformly varied. The chromatographic gel
should be mounted midway between the aligned pairs of electrodes with
equal but opposite charge. To monitor the applied electric potential, a
voltmeter whose input can be switched to any selected electrode pair is
used to measure the applied field intensity.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be described with reference to the accompanying
drawings, which form a part of this specification, and wherein:
FIG. 1 is a plot showing the change in the electric field as a function of
input voltage to the electrode array;
FIG. 2 is a graphical demonstration of the extensive variation in the
electric field intensity measured in a commercially available apparatus;
FIG. 3 shows autoradiograms of a series of transfers from gels containing
uniform suspensions of a radioactive macromolecule;
FIG. 4 shows computer simulations of asymmetric electrode arrays;
FIG. 5 shows the measured electric field in the transfer box of the instant
invention;
FIG. 6 presents computer generated models of the transfer box of the
instant invention;
FIG. 7 is an illustration of the utility of five independent, aligned
electrode pairs to produce both a very uniform field and gradient fields;
and
FIG. 8 is a demonstration of the utility of gradient field blotting.
DETAILED DESCRIPTION OF THE INVENTION
One parameter that directly affects the blotting process is the electric
field which serves as the motive force that drives the elution of
macromolecules. Variations in the electric field intensity cause
variability in the elution process. Therefore, it is important to subject
the macromolecules to the same field intensities to ensure their uniform
transfer. Therefore, a reliable blotting apparatus should generate
predictably uniform electric fields, i.e., fields without undesirable
detectable changes in field intensity. The standard procedure employed to
produce electric fields has been to use an array of platinum wire as the
electrode. In practice, arrays of various configurations have been
employed. The goal in routing the platinum wires comprising the electrode
arrays of opposite charge was to use as little material as possible while
at the same time attempting to route them so as to produce a uniform
field. No direct measurements of the field intensities produced by the
various configurations of electrode arrays have been reported. Aside from
the qualitative evidence (cited in the Prior Art section) for the
production of a uniform field with a particular electrode configuration
the uniformity of the field produced over the surface of the gel by other
electrode configurations is unknown.
To facilitate the design of an electrode array which would be efficient in
the use of the electrode material but would also produce the desired
uniform field at the frontal median plane a computer model was developed
which permits the analyses of the electric fields of a very large number
of simulated electrode arrays confined within a nonconductive box. Based
upon these data an electrode array has been designed that generates either
a highly uniform or a controlled gradient of field intensity. This
capability to generate a gradient can provide an efficient solution to a
major problem in macromolecule transfer that stems from the fact that
elution efficiency of a macromolecule is related inversely to molecular
weight. Since the gradient electric field can assume a variety of shapes
the gradient can be tailored to experimental needs.
A device was constructed to allow the direct measurement of an electric
field at various positions within the nonconductive box of a transfer
apparatus. The device consists of a probe and a mounting mechanism that
could be used to accurately position the probe vertically and
horizontally. The probe consists of two square pieces (0.25 cm.sup.2) of
15 mil platinum foil which are mounted parallel 1 cm. apart from each
other each on the end of a calibrated plexiglass rod. The squares of
platinum foil are connected via Teflon insulated wire to a voltmeter or
other measuring device such as a computer. The mounting mechanism consists
of a clamp which is mounted on a calibrated horizontal track and which
holds the probe. The horizontal position of the probe can be determined
manually or by a computer. The probe could be adjusted horizontally and
vertically in a reproducible manner anywhere within the nonconductive box
of the transfer apparatus. To test the efficacy of the device to measure
the change in the electric field as a function of the input voltage to the
electrode array, the series of measurements plotted in FIG. 1 were made.
The plot demonstrates that the potential difference measured between the
two squares of platinum foil was directly proportional to the electrical
input to the transfer apparatus. The measured voltages are considered to
reflect the average field intensity of the region of the nonconducting box
in which the probe is suspended.
Each electrical measurement and each transfer recited in this application
is conducted using 15.6 mM Tris, 120 mM glycine pH 8.3 as electrode buffer
and a power supply which provides 200 mA constant current. Measurements
are made while stirring the buffer thus reducing the effect of bubbles
that otherwise accumulate around the electrode wires.
To determine whether in fact uniform electric fields are produced, direct
measurements of field intensities generated by a variety of transfer
apparatus equipped with different configurations of electrode arrays (some
commercially available) were made. These measurements clearly indicate
that prior to this invention uniform electric fields were not produced at
the surface of the gel. An actual demonstration of the extensive variation
in the electric field intensity measured in a commercially available
transfer apparatus which utilized an asymmetric electrode array design of
three continuous horizontal lengths of platinum wire opposing two
staggered horizontal lengths in which all the vertical cross connections
are exposed is graphically reported in FIG. 2. FIG. 2 plots the volts
measured (electric field) vs. the distance from the surface of the buffer
toward the bottom of the box. The arrows in the accompanying electrode
diagrams show the position of the probe for each scan. All scans of the
field in the median frontal plane of the apparatus, run perpendicularly to
the electrodes, show single or complex peaks in the center of the box and
fall off unevenly at the top and bottom. Deviations in the field strength
are quite prominent even when the median frontal plane is scanned parallel
to the main lengths of this electrode array.
To prove that nonuniform electric fields do adversely affect both
qualitatively and quantitatively the transfer of macromolecules from
chromatographic gels, a series of transfers from gels containing a uniform
suspension of a radioactive macromolecule were performed (FIG. 3). These
polyacrylamide gels containing uniform suspensions of radioactive protein
were prepared by solubilizing 1-2 .mu.Ci.sup.125 I-labeled bovine serum
albumin in 0.9 ml of 10% aqueous SDS. This preparation was added to 89.1
ml of a 10% acrylamide in Tris-HCl buffer pH 8.8 and the resulting mixture
was polymerized to yield a 10% polyacrylamide gel containing 0.1% SDS and
a uniform suspension of radioactive protein. This is demonstrated by the
autoradiogram of such a gel in FIG. 3A. These gels were then used for
transfer to membrane filters. The autoradiograms of such filters
demonstrate the topography of the electric field generated in the
particular transfer apparatus being tested. A uniform electric field
produces uniformly exposed autoradiograms (FIG. 3C) whereas autoradiograms
produced from transfers conducted with nonuniform electric fields will
produce autoradiograms with varying degrees of exposure. FIG. 3B is the
autoradiograms of the blot obtained by transferring such a gel in the
apparatus analyzed in FIG. 2. The variations measured are reflected as
differential efficiency in transfer of .sup.125 I-labeled bovine serum
albumin. That such peaking in the center of the frontal plane appears to
be a characteristic trait to asymmetric arrays in general is demonstrated
in the computer simulations (FIG. 4).
Computer simulation of the electric fields generated by different designs
of electrode arrays is based on the mathematical expression which is
derived from basic principles of electrostatics. A computer model was
generated by defining the boundaries of a non-conductive box and the
locations and the coordinates of electrode elements within it. The
computer scans over a selected plane within the confines of the box and
calculates the potential generated by the electrode elements at each point
on the plane. This value is then quantized into 0.2 volt intervals. The
levels are then plotted onto the display at the specified coordinates. To
facilitate visual observation, the output is transposed into three grey
scale values and printed. The potential change from one grey scale value
to the next is 0.2 volts.
For each simulation the box dimensions and electrical input have been kept
constant. FIG. 4A-F depict the topography of the electric field in the
median transverse plane generated by pairs of electrodes within a box
where the anode consists of four vertical lengths and the cathode of
three, four, or five lengths as indicated. The positions of the electrode
elements can be determined from the reference points designated beyond the
walls of the box. The median of the plane is the plane where uniform
electric fields would be expected. Note that the four elements of the
cathode and the anode in FIG. 4C are staggered and compare this array to
the directly aligned elements as in the case for FIG. 4B. The increase in
nonuniformity with nonalignment is dramatic. The contribution to the field
from the connecting wires between the elements has been accounted for in
FIG. 4A-D. FIG. 4E-F depict the same electrode configuration as FIG. 4A-B
respectively, however, the connecting wires are insulated and do not
affect the electric field. It is apparent from FIG. 4 that the most
promising electrode configuration is the symmetrical 4.times.4 array.
Further fine tuning of this array is accomplished by placing the outer
electrode elements in a plane deeper into the box bringing them closer to
each other, thereby increasing the field directly between them. This has
the effect of broadening as well as extending considerably the region of
uniformity into other medial frontal planes.
Utilizing the results from the computer simulation of the electric field
from various configurations of electrode arrays, a transfer box equipped
with the symmetrical 4.times.4 configuration of an electrode array was
constructed and tested. The nonconductive box measures
10.times.16.times.24 cms. Four vertical lengths of a continuous platinum
wire opposed directly a second array of four vertical lengths identical to
the first, the electrodes in each pair being placed the same distance from
the sides of the box. The length of each vertical wire was 20.5 cm. gel.
The diameter of the wire was 14 mil. The two outer most lengths of each
electrode wire were positioned 0.3 cm closer to the median frontal plane
of the box than were the other two lengths which were directly against the
surface of the wall. The distance between each of the four vertical
lengths was 4 cms. All the cross connecting wires were insulated and thus
did not contribute to the electric field. The distance between the anode
and the cathode was 7.5 cm. FIG. 5 shows the measured electric field in
this transfer box. The field was measured across the frontal median plane
at 5 depths (every 4 cm; each depth is designated by a different symbol)
from the buffer surface. The plot demonstrates the uniformity of the
field. Computer generated "field maps" of such a box are presented in FIG.
6. (A is the frontal median plane, B is 0.5 cm off the median plane, C is
the median saggital plane, and D is the median transverse plane). The
uniformly darkened autoradiogram shown in FIG. 3C demonstrates that this
apparatus gives a uniform transfer of .sup.125 I-labeled bovine serum
albumin. This configuration of electrode wires ensures that a uniform
electric field can be reliably and reproducably generated. This ensures
that quantitative transfers of macromolecules of the same molecular weight
in every lane in a chromatographic gel will occur.
Measurements of the electric fields generated by the electrode array of the
same configuration but with insulated or uninsulated connecting wires
indicates clearly that wider, more symmetrical and more uniform fields are
obtained in the frontal central region of the box when the connecting
wires are insulated. Therefore, in the most preferred embodiment, these
connecting wires are omitted altogether. The electrode array consists of
an equal number of independent, aligned wire lengths, equally spaced from
one another mounted to the walls of the box. Whether the aligned wires are
to be mounted horizontally or vertically depends on the orientation of the
chromatographic gel. The spacing between the aligned independent wires
depends on the number of aligned wire pairs and the dimensions of the box.
In the most preferred embodiment the box is the same dimensions as
previously described. In the most preferred embodiment 5 independent pairs
of aligned electrodes are employed. The aligned pairs are equispaced from
each other and from the side walls of the box. When all five pairs of
electrodes are connected directly to the power supply they function as
would be expected of a continuous wire electrode with 5 horizontal lengths
and insulated connecting wires.
In addition to providing highly homogeneous fields this electrode
arrangment offers the possibility of generating controlled variable fields
of which linear gradient fields are of particular interest. Field
gradients may be generated in a variety of ways both mechanical and
electrical. Some mechanical means include tilting the electrode array such
that one end, for example the bottom end, has a closer distance from anode
to cathode than the other end. The bottom end would therefore have a
higher field intensity although it would be more difficult to adjust
reproducibly. Another mechanical means would employ a mask to be
sandwiched between the chromotographic gel-membrane filter assembly and an
electrode. The characteristics of this mask would be to have a variable
degree of electrical impedance from one end to the other. Such a
characteristic could be obtained either through the manipulation of the
mask composition or by varying the porosity of the material from one end
to the other. The net result of the mask placed in a homogeneous field
would be to generate a controlled gradient over the surface of the gel.
Such masks would be expensive to manufacture and separate masks would be
necessary to create different gradient fields. Yet another mechanical
means of producing a gradient would be a system to physically remove the
chromatographic gel-membrane filter assembly from the buffer with the
electric field at a controlled rate. The net effect of such a system would
be to cause a variable exposure time to the electric field. Thus the
chromatographic gel first removed at the top would receive the least
exposure while the bottom of the chromatographic gel would receive the
applied field for the full time duration. Though this system would work,
it is laden with problems, not the least of which is the drying of the gel
once it is removed from the buffer.
One electrical means of producing a gradient field would be to provide
independent power sources for each electrode pair. Such a system would
provide an easy means to manipulate the gradient but would be
prohibitively expensive to become practical. Another electrical means
would be to use a single power source but a system of independent voltage
or current regulators for each electrode pair. This would maintain the
ease of adjustment of the former method and would be less expensive. A
simpler method still is to provide either fixed resistors in series with
each electrode pair which could then be switched to establish different
gradients or a set of variable resistors for each electrode pair so that
the field could be continuously varied. The method employed in the most
preferred embodiment of the invention uses a combination of the above
methods a switch selects fixed resistors for preset gradients as well as
potentiometers for continuously variable gradients.
Whether setting variable gradients with potentiometers or observing the
effects of chromatographic gels in uniform fields, it is extremely useful
to monitor the applied potentials at each electrode pair. This capability
is realized in the preferred embodiment of this invention through the use
of a voltmeter, either housed within the control box or attached
externally, whose input can be switched to any selected electrode pair in
order to measure the applied field intensity. It is connected through the
selecting switch directly to the opposing pairs of electrodes after the
series resistance to the power supply.
FIG. 7 is a plot of the electric field measured from top to bottom at the
frontal median plane. FIG. 7 illustrates the utility of the five
independent, aligned electrode pairs to produce both a very uniform field
(solid dots) and gradient fields (solid squares, 65 V-15 V; open squares,
40 V-20 V).
To demonstrate the effect of the molecular weight of a macromolecule on its
transfer from a gel, .sup.125 I-labeled proteins of different molecular
weight were used as standards. These standards were separated on SDS/5-15%
polyacrylamide gradient gels and then blotted to nitrocellulose membrane
filters.
FIG. 8 clearly demonstrates the utility of gradient field blotting. As
noted above, the speed of electroelution of macromolecules at constant
current is inversely proportional to their molecular weight (the numbers
in FIG. 8 designate molecular mass in kilodaltons). If the voltage,
current and time conditions are selected so as to optimize the
electroelution of the lower molecular weight macromolecules, the larger
molecular weight components are retained by the gel. If the voltage,
current and time conditions are optimized to elute the high molecular
weight components, the lower molecular weight components are eluted so
rapidly that they cannot be retained by the immobilizing matrix. FIG. 8
compares gradient field electroelution (C) with the conventional method of
uniform field (25 volts, B) electroelution (A, is an autoradiogram of a
sample gel prior to transfer). It is apparent in the uniform field
transfer that high molecular weight components are dramatically diminished
in quantity. This is in contrast to the linear gradient field (15-65
volts) in which a more uniform and therefore a more quantitative transfer
of all of the components from the gel is obtained (compare A to C).
Therefore, gradient fields provide two very significant advantages. First,
there exists the possibility of preferentially accelerating the high
molecular weight proteins while slowly eluting the low molecular weight
proteins. Slow transfer of the smaller proteins is found to be
advantageous as it gives the macromolecules more time interact with the
matrix material thus limiting the extent to which they are blown through
the filter. Secondly, the gradient allows better use of common power
supplies. By redistributing the field strength, high potential
differences, e.g., 65 V, can be generated where needed and sufficiently
low, e.g., 15 V, provided at the lower end of the gradient while still
running the system with modest currents, e.g. 200 mA.
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