U.S. patent application number 13/286179 was filed with the patent office on 2012-06-07 for facile method and apparatus for the analysis of biological macromolecules in two dimensions using common and familiar electrophoresis formats.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to James T. Champagne.
Application Number | 20120138463 13/286179 |
Document ID | / |
Family ID | 40525074 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120138463 |
Kind Code |
A1 |
Champagne; James T. |
June 7, 2012 |
FACILE METHOD AND APPARATUS FOR THE ANALYSIS OF BIOLOGICAL
MACROMOLECULES IN TWO DIMENSIONS USING COMMON AND FAMILIAR
ELECTROPHORESIS FORMATS
Abstract
An ensemble of components and methods are disclosed for
utilizing two-dimensional electrophoresis in polyacrylamide or
related polymer gels using common, existing and familiar
electrophoresis formats and equipment. The disclosed invention
makes two-dimensional electrophoresis convenient and easy to use
for individuals already using vertical "mini-gel" type systems. The
invention discloses the combination of pre-cast disposable gels for
both first and second separation dimensions using novel support
devices and cassettes that simplify the difficult multiple sample
handling and processing steps Inherent in ordinary two-dimensional
electrophoresis methods. Devices and methods are disclosed in said
invention that provide exceptional gel to gel reproducibility.
Inventors: |
Champagne; James T.;
(Vashon, WA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
40525074 |
Appl. No.: |
13/286179 |
Filed: |
October 31, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12391809 |
Feb 24, 2009 |
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13286179 |
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09633172 |
Aug 4, 2000 |
7517442 |
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12391809 |
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60147490 |
Aug 9, 1999 |
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Current U.S.
Class: |
204/459 ;
204/610 |
Current CPC
Class: |
G01N 27/44782 20130101;
G01N 27/44795 20130101 |
Class at
Publication: |
204/459 ;
204/610 |
International
Class: |
B01D 57/02 20060101
B01D057/02 |
Claims
1. An apparatus for conducting two dimensional gel electrophoresis
of a protein sample comprising: an isoelectric focusing apparatus,
wherein said isoelectric focusing apparatus removably fits into an
isolating manifold apparatus designed to accept both said
isoelectric focusing apparatus and an apparatus for conducting the
second SDS-PAGE separation, and said isolating manifold fits into
any standard "mini-gel" electrophoresis apparatus; and an apparatus
for conducting the second SDS-PAGE separation step of a
two-dimensional gel electrophoresis; said apparatus for conducting
the second SDS-PAGE separation also removably fitting into said
isolating manifold apparatus.
2. An apparatus for conducting two dimensional gel electrophoresis
of a protein sample comprising: a) a first isoelectric focusing
apparatus for conducting a two-dimensional gel electrophoresis of a
sample further comprising: 1) a semi-rigid planar sheet having a
front and a back surface, a plurality of perforations disposed
longitudinally in one direction across said support; and also
having a plurality of registration holes disposed longitudinally in
the same direction and adjacent to said perforations across said
semi-rigid planar sheet; 2) a plurality of rectangular holes which
defining a casting space for a strip of first dimension
electrophoresis gel, and said casting space allowing electrical
conductivity for said first dimension electrophoresis gel through
said semi-rigid planar sheet to one or more electrolyte reservoirs;
said rectangular holes also disposed longitudinally in same
direction and adjacent to said perforations and registration holes
across said semi-rigid planar sheet; 3) at least one surface of
said semi-rigid planar sheet treated in such a manner to provide a
continuous or discontinuous hydrophilic reactive surface capable of
binding an electrophoresis gel polymer by covalently integrating
into said gel polymer matrix; 4) said perforations and registration
holes are sufficient to allow individual electrophoresis gel strips
set within said rectangular holes to be separated from the
remainder of the semi-rigid planar sheet; B) an isolating manifold
apparatus for conducting two-dimensional gel electrophoresis of a
protein sample further comprising: 1) a manifold constructed of
elastomeric materials, said manifold having a mounting face and an
opposite face and being capable of being fit into a support stand
or clamped to a standard gel electrophoresis clamp or glass or
plastic plate; 2) said manifold provided with a plurality of
parallel gel channels, on the opposite face of the manifold, said
gel channels having a top and bottom end, and wherein said channels
have approximately the same dimension and spacing as the
rectangular holes in the semi-rigid planar sheet of claim 1, and
said gel channels have a depth which defines a space for the first
dimension electrophoresis gel strips with a very small laminar void
running the entire length of the gel channel when said gel strips
are fully hydrated; 3) the opposite face of said manifold is
further provided with a sufficient number of registration tabs
between the gel channels along the perforation line that push
through the corresponding registration holes of said semi-rigid
support of claim 1, such that each gel channel is elastomerically
sealed from the others and from outside the manifold, and said
opposite face also having a ridge disposed around the perimeter of
said manifold, wherein said ridge contacts said semi-rigid support,
and holds said semi-rigid support tightly against the manifold face
by pressure; 4) in the opposite face of said manifold, at the
extreme top and bottom of each gel channel, a plurality of ports
are also provided which are approximately conical in shape and
roughly corresponding in dimensions to a disposable microtiter
pipette tip capable of holding volumes around 2 to 200 .mu.L, and
said ports are oriented so that when the manifold is mounted on a
support stand they are approximately vertical, and said ports
having a top and a bottom and the bottom of each port is connected
via a collapsible channel to a corresponding gel channel, and said
collapsible channel can be either molded into the manifold at the
time the manifold is constructed or can be cut through the port
after manifold construction, and said collapsible channel is
capable of remaining closed and forming a tight fluid seal between
the gel channel and the port until a pipette tip or similar means
is inserted into the port and allows the collapsible channel to
open and creates a fluid path between the port and the gel channel
until the tip is removed and said collapsible channel recloses; 5)
said manifold is capable of being attached to a buffer reservoir
means of the type commonly used in gel electrophoresis or mini-gel
electrophoresis, wherein the slot of the semi-rigid sheet of claim
1 can be aligned such that filling of the buffer reservoir means
allows electrical conduction of current through each gel strip in
said manifold for electrophoresis of samples; C) an apparatus for
conducting the second SDS-PAGE separation step of a two-dimensional
gel electrophoresis of a sample further comprising: 1) a molded
cassette capable of containing pre-cast gel electrophoresis media,
wherein said cassette has a top, two lateral sides and a bottom,
and said cassette also has a front plate located on the front of
the cassette, and a back plate located on the opposite side of the
cassette, both front and back plates having the general dimensions
that are known for existing "mini-gel" cassettes; 2) said front
plate being capable of accepting the mounting of a first dimension
gel strip within a horizontally oriented port or opening which is
molded into the surface of said front plate, and wherein said
horizontally oriented port or opening within the front plate is
capable of being temporarily sealed flush with the Inside face of
the front plate; 3) said cassette having sealing spacers provided
at the lateral sides and bottom which forms an interior laminar
space within the cassette between the front and back plates capable
of containing gel electrophoresis media in either a polymerized or
an unpolymerized state within said space, and wherein said cassette
is capable of maintaining this interior laminar space indefinitely;
4) said front and back plate further comprised of glass or molded
rigid plastic or elastomeric materials compatible with aqueous
solutions and said back plate also being capable of having the gel
facing surface of said back plate modified so that polymerized gel
electrophoresis media will chemically bond with the gel facing
surface of said back plate during or after polymerization; and 5)
the horizontally oriented port or opening within the front plate
further comprises a tight fitting elastomeric seal forming a
water-tight barrier in said horizontally oriented port or opening,
wherein said seal is comprised of molded rigid plastic or
elastomeric materials compatible with aqueous solutions, and said
seal provides the attachment of a first dimension gel strip with
its backing into a tight-fitting groove on the gel face of the
front plate, and said seal has a tab allowing for the removal of
the seal after the first dimension gel strip is in contact with the
second dimension gel media exposed through said horizontal opening
when said seal is in place.
3. A method for conducting two-dimensional gel electrophoresis on a
sample using the apparatus of claim 1 comprising the steps of: a)
partially dehydrated pre-cast polyacrylamide pH gradient gel strips
bound to a perforated semi-rigid support or backing are pressed or
mounted into molded channels on one side of said elastomeric
manifold so that each strip is sealed with a laminar space above
said gel, each laminar space is accessible for filling through
closable ports on top and bottom on the side of the elastomeric
manifold opposite from the strips; b) the elastomeric manifold is
mounted at an angle on a support stand wherein samples containing
proteins to be focused are loaded through the bottom ports through
opening the closable channels and filling the laminar space above
each strip, and the top ports are forced open so that the laminar
space can be vented; c) samples loaded are incubated for sufficient
time so as to re-hydrate into the isoelectric focusing gel media;
d) the manifold with up to eight attached sample isoelectric
focusing strips is mounted adjacent to the buffer core and sealed,
a buffer dam or a second manifold is installed on the opposite side
of said buffer core and said manifold or manifolds with said buffer
core is dropped into the lower buffer chamber; e) electrolytic
buffer solutions are added to the reservoirs to a level above
connecting slots punched through the perforated semi-rigid backing
at the top and bottom of each gel strip allowing an electrical path
to the isoelectric focusing gel from the reservoirs; f) the
apparatus is attached to a constant voltage power supply and run
for a sufficient period of time to migrate the samples into the gel
media and then the voltage is increased and continued until the
samples are sufficiently focused within the gel media; g) the
voltage is removed and said manifold is remounted on said support
stand, a re-equilibration solution is the added to the top ports of
the laminar spaces of each first dimension isoelectric focusing
strip and allowed to fill said spaces by gravity means, during
addition of solution, the lower ports of the laminar spaces are
kept open to provide venting; h) upon completion of filling of said
laminar spaces and waiting a sufficient time to denature the
samples in the gel media, said strips are removed from the manifold
and separated from each other by applying opposing force upon said
perforations in between each strip; i) a separated gel strip is
then pressed into an elastomeric seal means with the gel side on
the opposite side from the seal, and the strip and sealing means
are pressed into a slot in the lateral side of the second dimension
gel cassette so that the gel strip surface is in contact with
SDS-PAGE gel media of the second dimension gel cassette in the
slot; j) one or more second dimension gel cassettes are then placed
against a buffer core of the mini-gel electrophoresis apparatus and
kept in place by pressure through the elastomeric seal around the
bottom and both sides of the buffer core, and the buffer core and
lower buffer chamber are filled with electrolyte solution, and the
system is energized to apply either constant current or constant
voltage; k) after a period of time sufficient to migrate the
protein samples into the second dimension gel, the system is
de-energized and the first dimension strip and elastomeric seal are
removed from the second dimension gel cassette, the system is
energized for sufficient time to separate the sample proteins on
the second dimension gel; and l) upon completion, the system is
de-energized and the second dimension gel cassette is then removed
from the apparatus and separated from the buffer core where it is
further processed.
Description
[0001] This application claims priority from U.S. Provisional
Application No. 60/147,490 filed on Aug. 9, 1999.
TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF INVENTION
[0002] The invention relates to the production of acrylamide or
other gels for use in separations of proteins, nucleic acids or
other biological materials. The invention further relates to making
two-dimensional electrophoresis convenient and easy to use for
individuals already using vertical "mini-gel" type systems. The
invention discloses the combination of pre-cast disposable gels for
both first and second separation dimensions using novel support
devices and cassettes that simplify the difficult multiple sample
handling and processing steps inherent in ordinary two-dimensional
electrophoresis methods.
BACKGROUND OF THE INVENTION
[0003] Polyacrylamide gel electrophoresis has become one of the
most frequently used techniques for the separation of biological
macromolecules such as proteins, nucleic acids and polysaccharides.
There is already a wide variety of equipment and methods for many
types of high-resolution separation of these biological
macromolecules for both analytical and preparative purposes. Two of
the most widely used classes of separation methods involve: 1)
separating protein molecules according to molecular weight using
sodium dodecyl sulfate (SDS) denaturation, often referred to as
"SDS-PAGE", and 2) the separation of various types of amphoteric
molecules (i.e. molecules with more than one ionizable chemical
group) such as proteins using the principle of isoelectric focusing
(IEF) on stabilized pH gradients where molecules migrate to a
position in an electric field (the isoelectric point) where the pH
environment provides a net zero charge on such an amphoteric
molecule.
[0004] These two arts can be combined wherein a complex mixture of
amphoteric molecules are initially focused in a one dimensional pH
gradient means, such as a strip or tube of gel, to their component
isoelectric points. The focused amphoteric molecules in the one
dimensional pH gradient means are then subsequently separated by
molecular weight using SDS denaturing electrophoresis in an
orthogonal direction to the first dimension. This two-dimensional
separation of complex mixtures using polyacrylamide electrophoresis
is a powerful but difficult technique. It is often perceived as an
art to be reserved for specialists in a biological research
organizations who have cultivated the skills to create and
implement first dimensional pH gradients in tubes or strips and to
successfully equilibrate and transfer the resultant first
dimensional gel onto a second dimensional gel using difficult
manual transfer and alignment methods.
[0005] There is a great demand in everyday research, especially in
protein based research, for the high resolution and parallel
analysis that is afforded by this two-dimensional analytical
approach. If this technique could be made convenient and easy to
implement in a format that is familiar to the average laboratory
researcher, its use would greatly increase. This is especially true
for analysis of small regions of a larger two-dimensional gels. The
present invention discloses such a facile and convenient method of
implementing two-dimensional gel electrophoresis in the popular
research electrophoresis format known as a vertical "mini-gel".
SUMMARY OF THE INVENTION
[0006] The method provides for the pre-casting and partial
dehydration of a plurality of first dimensional pH gradient
isoelectric focusing gels cast onto a semi-rigid support in
separate parallel discontinuous strips. Each isoelectric focusing
gel is provided with an immobilized, and often a titrated
acrylamido-buffered, pH gradient or carrier ampholyte generated pH
gradient, on said semi-rigid support. The spacing of each of the
parallel discontinuous strips matches the standardized conventional
multi-well pipettor spacing of about 9 mm on center. In a preferred
embodiment of the present invention, the length of the strips is
about 8-12 cm, the width is about 3-5 mm and the thickness when
fully hydrated is about 0.2-1.0 mm.
[0007] Additionally, a molded tight fitting manifold with a
plurality of laminar spaces corresponding to each pre-cast strip is
provided for the absorption, re-hydration, sample loading and
subsequent isoelectric focusing of said strips. The laminar spaces
are preferably the same size and shape as the pre-cast isoelectric
focusing strips, with a depth slightly greater than the thickness
of the isoelectric focusing strip when fully hydrated. The
semi-rigid support backing of each strip fits into said manifold as
a unit such that each gel strip is sealed and isolated from the
other strips in a separate laminar space forming a gel
strip-manifold assembly.
[0008] The manifold also provides for a sealed laminar void space
running the length of each get strip adjacent to the face of each
partially dehydrated gel, which can be filled with a fluid medium.
The fluid medium is absorbed into the partially dehydrated get so
as to hydrate it. In a preferred embodiment, the manifold is molded
from elastomeric or semi-elastomeric polymers including but not
limited to polysiloxanes, polyisoprenes, polyisobutylenes and
polysulfides. Other elastomeric polymers which have physical
properties that provide for the easy and tight seal of the
semi-rigid support onto the manifold surface would suffice.
[0009] Each laminar void space, with its partially dehydrated
isoelectric focusing gel, forms a tight fitting sample loading
space adjacent to the gel. In a preferred embodiment, a reclosable,
opposing pair of ports are molded into the manifold at the top and
bottom of each laminar space. A fluid medium, which can contain
either buffer or the sample to be analyzed or separated, is
introduced into said laminar void space by opening the ports at the
top and bottom and filling the laminar void space through one port,
while allowing venting of air or buffer through the other port on
the opposite end of the laminar void space. When the fluid medium
is fully introduced, both top and bottom ports are reclosed. The
opening of these ports can be performed by any hollow tube like
dispensing means of proper diameter to fit within the port.
[0010] In a further preferred embodiment, the reclosable ports are
molded into the elastomeric manifold so they can be opened when a
pipette or other sample-loading device is used to introduce the
sample, or when used to vent entrained air from the port. The
opening is accomplished when the pipette or other sample-loading
device inserted, pushes the channel open. As said pipette or other
loading device is removed from the elastomeric manifold, the port
opening collapses against itself to form a tight fitting seal.
[0011] A diagonal support stand is provided in the present
invention to hold the gel strip-manifold assembly for the
introduction and incubation of said fluid media through the bottom
port of each laminar space and to allow for the venting of
entrained air through the top port. The 9-mm spacing provides for
convenient simultaneous sample loading with a commonly used 8 or 12
place pipettor (Gilson). A sample can then be introduced into the
isoelectric focusing gel by adsorption into and re-hydration of the
gel while being incubated in the said gel strip-manifold
assembly.
[0012] Once the sample is fully absorbed into the isoelectric
focusing gel strip, the gel strip-manifold assembly is flushed with
water to remove unabsorbed fluid. The manifold assembly is then
mounted into a standard vertical "mini-gel" electrophoresis
apparatus that has an upper and lower reservoir and a means of
introducing an electric field through the isoelectric focusing gel
from one end of the gel to the other. This vertical "mini-gel"
electrophoresis apparatus can be either be constructed for this
purpose or can be purchased at a variety of laboratory supply
houses (Bio-Rad, Hoefer Scientific, etc.) and adapted for use with
any of the conventional designs popular in research laboratories
using this electrophoresis format.
[0013] Following isoelectric focusing, said manifold is then used
with the vertical or diagonal support stand for in situ
equilibration of the completed first dimensional isoelectrically
focused gel, using buffer conditions required for the subsequent
second dimensional separation. Typically these second dimension
separation conditions require the rapid diffusion into the gel
strip of denaturing and reducing reagents, for example, but not
limited to sodium dodecyl sulfate detergent and dithiothreitol,
along with other reagents in one or more equilibration stages. Said
denaturing and reducing reagents are introduced through the same
reclosable ports in the same fashion as was used for introduction
of the sample prior to isoelectric focusing.
[0014] Pre-cast second dimension gradient gels of relatively
uniform dimension, concentration or pore size capable of being
contained in compatible molded cassettes are also provided in the
present invention. Said cassettes having a surface mounted
horizontal opening or port designed to accept a single fully
equilibrated isoelectric focusing strip near the top of said second
dimension gel. The horizontal opening or port is able to seal said
isoelectric focusing strip to the second dimension gel with flat
surface to flat surface contact. Said seal also isolates said strip
from the electroly1e chamber such that the second dimension gel
provides an electrical field vector which only runs vertically
through the mated first and second dimension gels and parallel to
the face of each gel in the manner usually afforded in vertical
"mini-gels".
[0015] In a preferred embodiment, a molded elastomeric seal
designed to accept the individual first dimension strip and isolate
it from the electrolyte reservoir provides this sealing function.
Such pre-cast second dimension cassettes may be adapted to function
in existing brands of `mini-gel apparatus or they may be designed
to function in a custom-built "mini-gel" apparatus.
[0016] It is often desirable to remove first dimension gel strips
or tubes from the second dimension gel after the sample has
completely migrated out of the first dimension gel and into the
second dimension gel. It is also desirable for the path of the
electrical conductivity to bypass the point of contact between the
first and second dimension gel. This bypass minimizes streaking
from poorly solubilized residual components on the surface of the
second dimension gel. Provision is provided for this in the way of
a tab or handle means on the horizontal port or seal such that the
seal and first dimensional strip can be removed following
de-energizing the apparatus. After de-energizing, the removal of
the first dimension gel is accomplished without disassembly or
emptying of the electrolyte solutions in the reservoirs. Following
removal of the seal and first dimension gel, the apparatus is
re-energized and the second dimension gel is run to completion with
the electrical path running through the now open horizontal port,
thus limiting the time without electric power to a minimum.
[0017] Therefore, it is an object of the present invention to
provide an apparatus which facilitates two-dimensional gel
electrophoresis.
[0018] Another object of the invention is to reduce handling and
movement of the gel, reduce operator exposure to buffers and
samples and improve test result reliability.
[0019] Another object of the invention is to provide an apparatus
to carry out horizontal as well as vertical movement of sample
molecules without touching the gel.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows a schematic representation in an exploded
isometric view of the various components of the method used for the
loading, sample absorption and subsequent re-equilibration of
samples to be analyzed using the preferred embodiment of the
present invention.
[0021] FIG. 2 shows a schematic representation of the details of a
semi-rigid support on which a plurality of parallel first
dimensional gel strips are cast with compositions allowing for pH
gradients to form and allowing for convenient further
processing.
[0022] FIG. 3A is a schematic cross sectional representation of the
details of an isolatni g manifold that allows said semi-rigid
support shown in FIG. 2 to form individual and isolated sample
loading laminar spaces when assembled with said isolating
manifold.
[0023] FIG. 3B shows the introduction of fluid media such as
samples or equilibration solutions through a reclosable port.
[0024] FIG. 3C shows the sealed absorption of the sample into gel
strips.
[0025] FIG. 3D is an isometric view of the second dimension gel
assembly.
[0026] FIG. 4A is a schematic cross-sectional representation
showing the arrangement of the various components used during the
first dimension.
[0027] FIG. 4B is a schematic cross-sectional representation
showing the arrangement of the various components used during the
second dimension.
[0028] FIG. 4C is a schematic cross-sectional representation
showing the arrangement of the various components used during the
second dimension with the top plate and seal in a exploded
view.
[0029] FIG. 5A is a schematic representation showing the mounting
of the pre-cast strips on the manifold.
[0030] FIG. 5B is a schematic representation showing the loading of
samples onto the gel strips on the manifold.
[0031] FIG. 5C is a schematic representation showing the incubation
of the samples on the manifold.
[0032] FIG. 5D is a schematic representation showing the attachment
of the manifold to the buffer core assembly.
[0033] FIG. 5E is a schematic representation showing the addition
of re-equilibration solution to the samples on the manifold.
[0034] FIG. 5F is a schematic representation showing the
disassembly/separation of the equilibrated gel strips in
preparation for second dimension electrophoresis and the separated
gel strips being placed in the second dimension electrophoresis
cassette.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION
[0035] Examples of two-dimensional electrophoresis procedures for
which the apparatus is well-suited are set forth in an article
entitled .cndot.Analytical and Micropreparative Two-Dimensional
Electrophoresis of Proteins" by M. G. Harrington, D. Gudeman, T.
Zewert, M. Yun, and L. Hood (METHODS: A Companion to Methods in
Enzymology, Volume 3, No. 2, Oct. pp. 98-108, 1991). The contents
of this reference along with all of the references cited in the
article are hereby incorporated by reference.
The First Dimensional Gel Strips and Semi-Rigid Support
[0036] In a preferred embodiment shown in FIG. 1, pre-cast
polyacrylamide or related pH gradient gel strips (1) are
polymerized on and covalently attached to a semi-rigid support (2)
such that the gel strips, which are typically about 3-5 mm wide,
are spaced at about 9 mm on center with around a 4-6 mm wide space
between them and a length of about 80-120 mm. The pre-cast gradient
gels can be cast in a discontinuous mold that allows for the
introduction of both uniform and gradient variable compositions
running the length of the gel. Said semi-rigid support (2), may
typically be composed of, but is not limited to, 100-200 micron
thick polyester, polymethylmethacrylate or polycarbonate film that
has been surface treated to provide a continuous or discontinuous
hydrophilic reactive surface that will bind the gel polymer by
covalently integrating into the gel matrix. An example of such film
is the polyacrylamide support film known as Gel Bond.TM..
[0037] Said semi-rigid support is provided, prior to casting, with
die cut perforations (3) and registration holes (4) between each
parallel gel strip such that individual strips can be tightly held
yet quickly separated from one another by snapping or cutting them
off. Said semi-rigid support is also provided with a die cut
rectangular hole (5) preferably about 0.5 mm high by the width of
the subsequent gel (around 3-5 mm) and located at the extreme top
and bottom ends of the gel casting space. These holes provide an
electrical connection for the first dimension gel strip through
said semi-rigid support to the electrolyte reservoirs without
substantial loss of gel support or dead space on the gel.
[0038] The polyacrylamide or other gel used in the first
dimensional strips can vary in both concentration and composition
to provide a wide variety of pH gradient ranges, and denaturing or
non-denaturing conditions. Some of these compositions can include
but are not limited to chaotropic reagent denaturation such as 6-9M
urea, chemically reduced conditions, narrow or broad range carrier
ampholytes or immobilized acrylamido pH gradients co-polymerized
into the gel.
[0039] It is desirable to provide the average laboratory researcher
with many overlapping pH ranges and molecular weight ranges that
allow them to choose a region that contains two-dimensionally
separated proteins of predetermined individual interest. This
"window" on one region of the overall two-dimensional grid
simplifies the analysis when one is not necessarily interested in
exploration of unknown effects and unknown target molecules.
The Isolating Manifold
[0040] A preferred embodiment shown in FIG. 2 comprises a manifold
(6) molded of elastomeric materials such that it can be stretched
and mounted onto a more rigid support stand (7), standard gel
clamping device or glass plate. Said manifold (6) is provided with
a plurality of parallel rectangular channels (8) at around 9 mm on
center on the face that is opposite of the mounting face. These
rectangular channels are the width and length of the corresponding
first dimension gels (1) and have a depth that provides for a very
small laminar void space running the length of the gel when the gel
is fully hydrated and at its standard thickness. In a preferred
embodiment, this laminar void space would be in the order of about
10% to 20% of the standard thickness of the gel.
[0041] The face of the manifold (6) with said channels (8) is
further provided with registration tabs (9) between the individual
gels along the perforation line that push through corresponding die
cut registration holes (4) in the semi-rigid support (2), and also
have a perimeter ridge to snap and hold the semi-rigid support (2)
tightly in place against the manifold (6). The number of
registration tabs is as required to completely seal each channel
from the others and from the outside, taking advantage of the
properties of elastomeric polymers to seal against smooth surfaces
when slight pressure is applied perpendicular to the surface.
[0042] On the opposite side of the elastomeric manifold, at the
extreme top and bottom of each gel channel (8), a conical shaped
venting or loading port (10) is molded into the manifold (6)
corresponding to the shape of a disposable pipette tip of the type
commonly found in biological research laboratories (such as
Gilson.RTM., or Pipetteman.RTM.)(FIG. 3A). At the bottom of said
venting or loading ports (10) is a collapsible channel (11) that
connects each conical port to the face of the corresponding void
space away from the gel when the semi-rigid support and gel is in
place. Said collapsible channel (11) is molded into the elastomeric
manifold at the time of it polymerization or it is cut through
between the conical port (10) and the void space after
polymerization of the elastomeric manifold. In a preferred
embodiment, the cross section of said connecting channel (11) is
very flat (typically about 1-2 mm wide.times.0.1 mm or less high).
Said channel (11) is also designed so as to be able to be pushed
open when a disposable plastic pipette tip is Inserted into the
conical port (FIG. 38) thereby affording a fluid path into the
connected void space on the opposite side of the manifold. This
connecting channel is further designed to collapse FIG. 3C and
reform a tight fluid seal between the conical port and the void
space when the disposable plastic pipette tip is removed.
[0043] Said conical ports (10) are oriented to be vertical or
nearly vertical when the gel strip manifold assembly is mounted on
the support stand used for loading and incubating the samples. This
orientation allows for loading of the samples through the bottom
conical port on each gel channel by displacement means while
pipette tips inserted in the top conical port on each gel channel
allow for venting of entrained air or other lower density fluid
than the fluid being introduced. In a preferred embodiment, a
support stand (7) for the manifold supports the manifold (6) at a
diagonal angle with the conical ports being vertical (FIG. 2).
[0044] The fully loaded gel strip manifold assembly is mounted and
sealed against a buffer core of the type usually found in existing
mini-gel electrophoresis apparatus using the standard method
provided for each brand of mini-gel electrophoresis apparatus (FIG.
4A). The slot (5) that passes through the semi-rigid backing (2) is
aligned such that filling of the upper and lower electrolyte
reservoirs makes electrical connections between the electrodes and
the gel strip (1) inside the manifold assembly for subsequent
isoelectric focusing of the gel strips.
The Second Dimension Gel Cassette and Gel Strip Transfer
[0045] In a preferred embodiment, a molded cassette (13) is
provided to contain a pre-cast second dimension gel which is either
a uniform concentration, or pore size gradient gel (14) (FIG. 30).
Said cassette (13) is constructed of a front plate (15) that
accepts the mounting of the fully focused first dimension gel strip
(1) in a horizontal opening or port (16) molded into said front
plate (15) of the said cassette and a back plate (17) located on
the opposite face of said cassette. The said cassette is of the
general size and design of existing pre-cast gel cassettes
available from various sources that are referred to as "mini-gel
cassettes". Said existing cassettes are typically around 8-10
centimeters high by about 10 centimeters wide and typically contain
the pre-cast gel during polymerization by the manufacturer or end
user and during subsequent use by the end user.
[0046] Said cassette (13) may be used to contain a polyacrylamide
or related liquid gel solution during polymerization in which case
the horizontal opening or port is temporarily sealed flush with the
inside face of the front plate (15) in order to mold a uniform
thickness gel. Sealing spacers (20) are also provided at the sides
and bottom of the cassette to contain the gel during
polymerization.
[0047] Alternatively, in another preferred embodiment, said
cassette (13) may, following polymerization in a separate molding
manifold, subsequently enclose the pre-cast gel (14). Said
enclosure of the pre-cast gel is accomplished by assembling the
front plate (15) against the back plate (17) such that the pre-cast
gel is accommodated in a correctly sized laminar space between the
two plates that contains the pre-cast gel (14) without air spaces
between the gel and the front plate (15) or back plate-(17). The
enclosure process may also seal other faces of the pre-cast gel
(13) and hold the parts of the cassette together.
[0048] In this same preferred embodiment of the invention, the back
plate (17) of said cassette (13) may be composed of or chemically
modified on its gel facing surface with reactive moieties that will
chemically bond the subsequently polymerized gel to said back plate
(17) during or after said polymerization. In this case, the back
plate (17) is mounted In said separate molding manifold prior to
pre-casting the gel. The back plate (17) with the attached gel is
then assembled, after pre-casting, with the front plate (15) to
form the pre-cast gel cassette (13).
[0049] Said back plate (17) of said cassette (13) can be composed
of glass or plastic materials compatible with the aqueous solutions
used for polymerization and use of polyacrylamide gels. The front
plate (15) can be composed of molded rigid plastic or elastomeric
plastic materials suitable for molding the horizontal opening and
compatible with said aqueous solutions.
[0050] The horizontal opening or port (16) molded into said front
plate (15) is provided with a tight fitting elastomeric seal (18)
that forms a water tight barrier in said horizontal opening (16).
This elastomeric seal may be composed of the same materials as the
isolating manifold (6). Said elastomeric seal provides for the
attachment of the fully focused first dimension isoelectric
focusing gel strip (1) on backing (2) in a tight fitting groove
(19) on the side facing the second dimensional pre-cast gel (14).
In a preferred embodiment, the elastomeric seal (18) is provided
with a tab (23) for easy removal while submerged in place in the
horizontal opening (16). The alignment of said first dimension gel
strip (1) Is such that said gel strip is placed in direct face to
face contact with the part of the second dimension pre-cast gel
(14) that is exposed through the horizontal opening (16) when the
elastomeric seal is tightly fitted Into said horizontal opening
(16).
[0051] The mounting of the elastomeric seal (18) and attached gel
strip (1) with backing (2) into the horizontal opening (16) is
performed in a manner that places said gel strip Into direct
contact with the second dimension pre-cast gel without entrapping
or entraining air bubbles. Typically, this can be accomplished by
placing one side of the flexible assembly into the opening first
and pushing the remainder of the assembly down with a sliding
motion from the starting end to the opposite end.
[0052] Said cassette (13), with the first dimension gel strip (1)
and elastomeric seal (18) assembly in place in the horizontal
opening or port (16), is subsequently mounted and sealed against a
buffer core of the type usually found in existing mini-gel
electrophoresis apparatus using the standard method provided for
each brand of gel electrophoresis apparatus. This assembly is
lowered into the lower buffer chamber of said electrophoresis
apparatus and SDS-PAGE electrolyte solutions are filled into the
reservoirs. As illustrated in FIG. 4B, a lid, electrodes and a
power source are attached to the electrophoresis apparatus and the
second dimension gel is run so as to transfer the sample proteins
from the first dimension gel strip (1) into the second dimension
pre-cast gel (14) adjacent to the horizontal opening (16).
[0053] A current path is provided, in the typical fashion for
"mini-gels", from the upper electrolyte reservoir, through the
exposed and submerged top edge (21) of the second dimension
pre-cast gel (14) and out the exposed bottom edge (22) of said gel
into the lower electrolyte reservoir (FIG. 4B). The lines of
electrical force pass into the electrically continuous first
dimension gel strip and electrophoretically mobilize the sample
proteins in the strip. As shown in FIG. 4C, in a preferred
embodiment, said gel strip (1) and the elastomeric seal (18) can
subsequently be removed by pulling on the tab (23) after complete
sample transfer from the gel strip (1) into the second dimension
pre-cast gel (14) and following de-energization of the
apparatus.
[0054] The removal of said gel strip (1) and elastomeric seal (18)
allows the current path to run from the upper electrolyte reservoir
through the now open port (16) bypassing the location of transfer
and into the gel. The lower electrolyte reservoir is connected
through the bottom edge of the gel (22) as before. This arrangement
provides for Jess streaking and interference from residual
non-transferred sample components located on the surface of the
second dimension pre-cast gel (14) and is generally known to be
beneficial.
[0055] In an alternate embodiment of the present invention, the
front plate (15) is assembled onto a separately polymerized
pre-cast gel (14) to form the cassette (13), and said front plate
(15) is constructed of elastomeric materials of the same type as
described for the isolating manifold (6), the use of the
elastomeric seal (18) as a means of aligning the first dimension
gel strip (1) can be eliminated.
[0056] In this alternate embodiment, the front plate (15) has the
horizontal opening and a groove to accept the semi-rigid gel strip
backing plate (2) both molded into the front face of the front
plate opposite the side that faces the second dimension pre-cast
gel (14). The fully focused and separated first dimension gel strip
(1) on its semi-rigid backing (2) is mounted directly in the
horizontal opening or port (16) in the same fashion as the
elastomeric seal is mounted in the previous embodiment of the
invention so as to prevent entrapment or entrainment of air
bubbles.
[0057] The following is a step by step example of how the preferred
embodiment of the present invention can be used.
Example 1
Step 1: Mount Dehydrated Strips on Manifold
[0058] FIG. 5A shows partially dehydrated pre-cast polyacrylamide
pH gradient gel strips (1), bound to a perforated plastic backing
(2) at 9.0 millimeter spacing, are pressed into molded channels (8)
on one side of an elastomeric manifold (6) so that each strip is
sealed with a laminae space above the gel. Each laminar space is
accessible for filling through two normally closed diagonal ports,
top and bottom on the opposite side of the elastomeric manifold
from the strips.
Step 2: Load Samples
[0059] FIG. 5B shows how the elastomeric manifold (6) is mounted at
an angle on the diagonal support stand (7). Samples containing
proteins to be focused are loaded through the bottom ports (10),
pushing open the collapsible channels (11) and filling the laminae
space above each strip. Sample loading is most easily accomplished
using an 8-place pipettor. Sets of disposable pipette tips are
first inserted in the top ports. The tips hold the top port (10)
open through the normally closed collapsible channels (11) in order
to vent the laminar space.
Step 3: Incubate to Absorb Sample into Gel
[0060] FIG. 5C shows that as the pipette tips are removed, the
ports (10) in the elastomeric material reclose the collapsible
channels (11) to seal the laminar space containing each sample.
During an incubation lasting several hours, the samples will
re-hydrate into the isoelectric focusing strips and be absorbed
along with other reagents such as detergents and urea. After
Rehydration is complete, the entire assembly is ready for the first
dimension isoelectric focusing step.
Step 4: Place Manifold Against the Buffer Core
[0061] Any excess fluid in the laminar space is removed with a
pipette. The manifold (6), with as many as eight attached sample
loaded isoelectric focusing strips, is mounted on the "mini-gel"
buffer core and sealed. A buffer dam or second manifold is
installed on the opposite side and the assembly is dropped into the
lower buffer chamber of the apparatus. Electrolyte is added to the
reservoirs to levels above the connecting slots (5) that have been
punched through the perforated plastic backing (2) at the top and
the bottom of each gel strip. This conductivity path allows the
isoelectric focusing gel to be electrically connected to the
reservoirs.
Step 5: Run First Dimension Gel
[0062] The cell is attached to a constant voltage power supply and
run for about an hour at low voltage to load the samples into the
focusing gel. The voltage is then increased to complete isoelectric
focusing over a convenient run time.
Step 6: Re-Equilibrate Strips for SDS PAGE
[0063] FIG. 5E shows that at the end of the first dimension
isoelectric focusing run, the manifold (6) is either quickly frozen
for latter use or remounted on the support stand. Re-equilibration
solution containing SDS detergent, and DTT is filled into the
laminar spaces to denature and reduce the proteins in the strip.
The reagents flow by gravity from the upper ports to the lower
ports (10). They may be refilled as needed. The SDS denatured gels
are then removed from the manifold ready to separate into
individual strips and apply to the second dimension gel.
Step 7: Separate the Gel Strips (Steps 7-10 are Depicted in FIG.
5F)
[0064] FIG. 5F shows that re-equilibrated first dimension gel
strips (1) are quickly snapped apart and each is press mounted in a
molded groove of the elastomeric seal (18) with the gel side of the
strip facing out.
Step 8: Apply to Second Dimension
[0065] The first dimension strip (1) and seal (18) are carefully
pressed into a slot (16) in the side of the second dimension gel
cassette (13) by starting at one end so as to make face to face
contact with the exposed surface of the second dimension
polyacrylamide gel in the cassette slot.
Step 9: Transfer Proteins
[0066] The gel cassettes are pressed against the buffer core,
sealed and dropped into the lower buffer chamber. The buffer core
and lower buffer chamber are filled with electrolyte solution and
the system is energized during the migration of proteins out of the
strip.
Step 10: Complete Second Dimension Separation
[0067] After all the focused proteins have migrated into the second
dimension gel, the power is interrupted and the first dimension
strip and elastomeric seal are removed. The power is reset for the
remainder of the run with the current path now running through the
slot in the side of the cassette. Following completion of the
second dimension, the gels are removed and stained or
transblotted.
[0068] Although the invention has been described above in detail
for the purpose of illustration, it is understood that numerous
variations and alterations may be made by the skilled artisan
without departing from the spirit and scope of the invention
defined by the following claims.
* * * * *