U.S. patent application number 12/958244 was filed with the patent office on 2011-06-09 for solution phase electrophoresis device, components, and methods.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Joseph W. AMSHEY, Thomas R. JACKSON, Michael THACKER, Timothy V. UPDYKE.
Application Number | 20110132763 12/958244 |
Document ID | / |
Family ID | 33479254 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110132763 |
Kind Code |
A1 |
AMSHEY; Joseph W. ; et
al. |
June 9, 2011 |
SOLUTION PHASE ELECTROPHORESIS DEVICE, COMPONENTS, AND METHODS
Abstract
A device for fluid phase electrophoresis, particularly solution
phase isoelectric focusing, components thereof, and methods for use
are presented.
Inventors: |
AMSHEY; Joseph W.;
(Encinitas, CA) ; UPDYKE; Timothy V.; (Temecula,
CA) ; JACKSON; Thomas R.; (La Jolla, CA) ;
THACKER; Michael; (San Diego, CA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
33479254 |
Appl. No.: |
12/958244 |
Filed: |
December 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10843526 |
May 10, 2004 |
7850835 |
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12958244 |
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60469538 |
May 9, 2003 |
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60471390 |
May 16, 2003 |
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Current U.S.
Class: |
204/644 ;
204/600 |
Current CPC
Class: |
G01N 27/44795 20130101;
G01N 27/44704 20130101 |
Class at
Publication: |
204/644 ;
204/600 |
International
Class: |
G01N 27/447 20060101
G01N027/447; C25B 9/00 20060101 C25B009/00 |
Claims
1. A device for solution phase electrophoretic separation of
analytes within a sample, comprising: an anode; a cathode; a
chamber stack disposed between said anode and said cathode; and
chamber stacking means, wherein said chamber stack comprises a
plurality of detachably mated sample chambers aligned along the
electrical axis between said anode and cathode, the lumens of said
coaxially aligned sample chambers being collectively capable of
defining an electrically conductive fluid column therethrough;
wherein said chamber stack further comprises a plurality of
junctional partitions, each of said partitions positioned at a
different one of the junctions between adjacent sample chambers,
said partitions being permeable to ions and to at least a plurality
of the analytes in said sample; and wherein said chamber stacking
means is disposed completely external to said chamber stack.
2. The device of claim 1, wherein the lumen of each of said sample
chambers is rotationally nonsymmetric within the vertical plane
orthogonal to the electrical axis.
3. The device of claim 1, wherein the lumen of each of said sample
chambers is nonsymmetric across the horizontal plane.
4. The device of claim 3, wherein the lumen of each of said sample
chambers is ovoid within the vertical plane orthogonal to the
electrical axis.
5. The device of claim 4, wherein the lumen of each of said sample
chambers has a more acute radius of curvature at the bottom than at
the top.
6. The device of claim 1, wherein said chamber lumen has a volume
of less than about 2 ml.
7. The device of claim 6, wherein said chamber lumen has a volume
of less than about 1.5 ml.
8. The device of claim 7, wherein said chamber lumen has a volume
of less than about 1.0 ml.
9. The device of claim 8, wherein said chamber lumen has a volume
of less than about 750 .mu.l.
10. The device of claim 9, wherein said chamber lumen has a volume
of about 600-700 .mu.l.
11. The device of claim 1, wherein each said sample chamber further
comprises at least one port, said at least one port capable of
fluidly connecting said chamber lumen with the exterior of said
chamber when said chamber is mated within the chamber stack.
12. The device of claim 11, wherein each said sample chamber lumen
is ovoid with a more acute radius of curvature at the bottom than
at the top, and at least one of said sample chamber ports is at the
top of each said chamber.
13. The device of claim 12, wherein each said sample chamber port
is inwardly tapered.
14. The device of claim 1, wherein said sample chambers are capable
of mating solely by application of axially directed force.
15. The device of claim 14, wherein said mated chambers are
incapable of interchamber rotation.
16. The device of claim 15, wherein the mating of said chambers
positions all said chamber ports upward.
17. The device of claim 1, wherein each said partition has the
shape of a sample chamber lumen.
18. The device of claim 17, wherein each said partition has an
ovoid shape.
19. The device of claim 1, wherein at least a plurality of said
partitions have a fixed pH.
20. The device of claim 19, wherein each of said plurality has a
different fixed pH.
21-96. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of devices,
components, and methods for electrophoresis, particularly
isoelectric focusing, more particularly solution phase isoelectric
focusing (IEF).
BACKGROUND OF THE INVENTION
[0002] The complexity of eukaryotic proteomes--that is, the total
number of distinct protein species present concurrently in a
eukaryotic cell, including alternatively spliced isoforms and
variants differing in post-translational modification--typically
exceeds the resolving capacity of current analytical
techniques.
[0003] For example, the number of distinct protein species in
eukaryotic cells typically far exceeds the spatial resolution of
two-dimensional polyacrylamide gel electrophoresis (2D PAGE) gels,
with large numbers of distinct protein species appearing to
comigrate. The limited spatial resolution in turn constrains the
dynamic detection range of the technique: efforts to observe low
abundance species by increasing the initial protein load lead to
increased obscuration by high abundance species.
[0004] Recently, efforts have been made to increase resolution of
such protein analytical techniques by prefractionating the protein
mixture prior to analysis. In one approach, complex mixtures are
prefractionated using solution phase isoelectric focusing, yielding
fractions having distinct pI ranges that can thereafter be
separately analyzed by 2D PAGE with increased resolution. See Zuo
and Speicher, Anal. Biochem. 284:266-278 (2000); Zuo et al.,
Electrophoresis 22:1603-1615 (2001); Zuo and Speicher, Proteomics
2:58-(2002); Ali-Khan et al., Current Protocols in Protein Science
22.1:1-19 (2002); Zuo et al., J. Chromatography B 782:253-265
(2002); and Wistar Institute, WO 01/75432, the disclosures of which
are incorporated herein by reference in their entireties. See also
Tan et al., Electrophoresis 23:3599-3607 (2002); WO 01/36449; WO
00/17631; and Righetti et al., J. Chromatography 475:293-309
(1989).
[0005] A number of devices capable of solution phase isoelectric
focusing are available commercially. None of the devices, however,
provides particularly convenient solution phase IEF
prefractionation of small volume protein samples with a simple
device in a format that readily interfaces with subsequent
analytical techniques such as 2D PAGE.
[0006] There thus exists a continuing need in the art for devices,
components, and methods for solution phase electrophoresis,
particularly solution phase isoelectric focusing.
SUMMARY OF THE INVENTION
[0007] The present invention satisfies these and other needs in the
art by providing a solution phase isoelectric focusing device that
is assembled by simply inserting components (e.g., chambers) into a
loading tube and holding them in place with a single screw cap. The
screw cap is tightened to ensure leak-proof seals between the
chambers and other component parts. The device is easy to
disassemble by simply sliding the chambers and other components out
of the loading tube. In preferred embodiments, air bubbles are
avoided in sample chambers by using optional specially shaped
openings and cap seals to seal the chambers after loading and/or
during electrophoresis or subsequent manipulations.
[0008] Generally, in IEF applications, the device uses fixed pH
membrane disks. Such disks can have a shape that limits the
electrodecantation of proteins. The shape of the membrane disks can
also be used to properly orientate a disk in position between two
chambers, thus minimizing or eliminating leaks that arise from
poorly orientated disks. Disk shapes that have these and other
desirable aspects are disclosed herein.
[0009] In one aspect, the invention relates to devices for IEF. In
some embodiments, the device does not require means for sample
recirculation, mixing or other agitation within its chambers during
the electrophoretic separation. In other embodiments, however, the
device is positioned on a rocker platform or a rotary platform to
move the entire device during operation, thus effecting sample
agitation.
[0010] In another embodiment, the invention relates to IEF devices
having a loading tube into which a plurality of sample chambers is
loaded. The tube aligns the sample chambers in a coaxial
orientation during assembly and operation. The chambers may be
compressed using one or more actuators, such as a screw cap at one
end of the loading tube.
[0011] Thus, in some embodiments, sample chambers are held tightly
in position by a screw cap at one end of the loading tube. The
screw cap compresses the chambers against one another as it is
tightened. Sealing O-rings between the chambers compress under the
pressure to form leak-proof seals.
[0012] In further embodiments, an anode end piece is introduced to
the loading tube before the first sample chamber to facilitate
subsequent disassembly of the device. The anode end piece has a
protrusion that slides along a channel in the loading tube,
facilitating removal of chambers from the loading tube.
[0013] In yet further embodiments, the electrode wires, which are
submerged in electrode buffer during use, are housed in
circumferential detents in cylindrical electrode plugs. The
recessed electrode wires are protected from damage during use and
during cleaning. The electrode plugs are optionally removable from
the device for cleaning, repair or replacement. Preferably, the
electrodes provide for a more consistent electrical field
regardless of support structure rotational orientation.
[0014] In yet further embodiments, the sample chambers have loading
ports and corresponding cap seals having a design such that air is
not trapped in sample chambers when they are sealed. A cap seal of
the invention does not completely block the fill port until the cap
seal is fully inserted, so that no air is trapped in closing the
sample chambers.
[0015] Moreover, the present invention relates generally to methods
and designs for sealing sample chambers without trapping air
bubbles, wherein air is displaced from sample chambers by slightly
over-filling the sample chamber with a supernatant fluid, and the
excess of such fluid is displaced as the cap seal is inserted into
the loading port. Such methods and cap seals can be used in other
applications, both in those involving aqueous solutions, as well as
other liquids. Other liquids include without limitation organic
solvents (e.g., benzene, other aromatic hydrocarbons, chlorinated
hydrocarbons, hexane, dichloromethane, alcohols, ketones, ethers,
amines, esters, petroleum products (oil, gas, etc.), paints,
primers, sealants, liquefied gases (e.g., liquid oxygen, liquid
nitrogen, etc.), culture media (i.e., for anaerobic bacteria) and
the like. In general, this aspect of the invention applies to any
liquid container the use of which would be enhanced by reduced
contact with air and/or fewer air bubbles.
[0016] In another aspect, the present invention relates to
immobilized buffer membranes for use in isoelectric focusing in one
or more other embodiments of the invention.
[0017] In a further aspect, the invention relates to kits for
solution IEF comprising the disks of the invention. Optionally,
such kits further comprise one or more buffers, one or more sets of
instruction, one or more protein standards, and/or one or more
control samples.
[0018] In a further aspect, the invention relates to methods of
separating, fractionating and characterizing proteins and other
biomolecules using a device of the invention. Such processes
involve electrophoresis, including without limitation isoelectric
focusing (IEF).
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] These and other objects and advantages of the present
invention will be apparent upon consideration of the following
detailed description, taken in conjunction with the accompanying
drawings, in which like graphical representations and like
characters refer to like structures throughout, and in which the
terms "proximal" and "distal" refer to the position of a component
relative to the anode when the device of the present invention is
assembled and ready for use.
[0020] FIG. 1 is a partially exploded proximal perspective view
(from above) of one embodiment of the device of the present
invention.
[0021] FIG. 2A is a partially exploded proximal perspective view
(from above) of one embodiment of the device of the present
invention, without the lid shown in FIG. 1.
[0022] FIG. 2B is a proximal perspective view (from above) of the
components shown in FIG. 2A, fully assembled. The axis of the
electrical field is indicated by a dashed line.
[0023] FIG. 3A is an exploded proximal perspective view (from
above) of the cathode buffer chamber and the spill trough of an
embodiment of the device of the present invention, with the cathode
electrode plug disassembled from the cathode buffer chamber.
[0024] FIG. 3B is a proximal perspective view (from above) of the
cathode buffer chamber and the spill trough of an embodiment of the
device of the present invention, with the cathode electrode plug
operationally engaged in the cathode port.
[0025] FIG. 4A is a distal perspective view (from above) of the
anode buffer chamber and the loading tube of an embodiment of the
device of the present invention, with the anode electrode plug
disassembled from the anode port.
[0026] FIG. 4B is a distal perspective view (from above) of the
anode buffer chamber and the loading tube of an embodiment of the
device of the present invention, with the anode electrode plug
operationally engaged in the anode buffer chamber.
[0027] FIG. 5A is a partially exploded proximal perspective view
(from above) of the anode buffer chamber, the loading tube, and the
chamber stack of an embodiment of the present invention. In this
exemplary view, sealing O-ring 29, immobilized buffer disk (IBD)
28, and spacer 30 are all shown positioned between two sample
chambers 2; in typical embodiments, however, IBD 28 and spacer 30
are alternatives, and only one of the two is positioned with an
O-ring between adjacent sample chambers.
[0028] FIG. 5B is a partially exploded distal perspective view
(from above) of the same components as in FIG. 5A.
[0029] FIG. 5C is a distal perspective view (from above) of the
anode buffer chamber, the loading tube, and several sample chambers
and associated components of an embodiment of the present
invention, assembled, but without the screw cap.
[0030] FIG. 5D is a distal perspective view (from above) of the
components shown in FIG. 5C, with the screw cap operationally
engaged.
[0031] FIG. 6A is an exploded proximal perspective view (from
above) of one embodiment of a sample chamber of the present
invention, with fill port, a cap seal for the fill port, a sealing
O-ring, and a spacer.
[0032] FIG. 6B is an exploded distal perspective view (from above)
of one embodiment of a sample chamber according to the present
invention, showing the use of an IBD as an alternative to use of a
spacer.
[0033] FIG. 6C is a proximal perspective view (from above) of an
embodiment of a sample chamber of the present invention showing a
cap seal engaged in the chamber fill port, and a sealing O-ring,
assembled.
[0034] FIG. 7A is an orthogonal midsectional view of an embodiment
of a sample chamber of the present invention, viewed along line A-A
of FIG. 6C, showing the fill port.
[0035] FIG. 7B is an orthogonal midsectional view of the sample
chamber of FIG. 7A, showing a cap seal operationally inserted into
the fill port.
[0036] FIG. 7C is an orthogonal midsectional view of an alternative
embodiment of a sample chamber of the present invention having a
tapered fill port, viewed along line A-A of FIG. 6C.
[0037] FIG. 7D is an orthogonal midsectional view of the sample
chamber of FIG. 7C, showing an alternative cap seal, properly
dimensioned to seal the tapered fill port, engaged within the fill
port.
[0038] FIG. 7E is a bottom orthogonal view of an embodiment of the
cap seal of FIG. 7D.
[0039] FIG. 8 is a distal perspective view (from above) of an
embodiment of a cathode end piece of the present invention; the end
piece interfaces the distal-most sample chamber of the chamber
stack with the screw cap (not shown).
[0040] FIG. 9A is a proximal perspective view (from above) of a lid
and associated anode and cathode cables, according to one
embodiment of the present invention. Also shown are the anode and
cathode tab slots of the lid.
[0041] FIG. 9B is a proximal perspective view (from above) of the
lid and associated electrode cables of FIG. 9A, assembled with
additional components of an embodiment of a device according to the
present invention. In the embodiment shown, the cathode tab slot of
the lid is shaped to receive only the cathode tab of the device,
thus preventing the lid from being assembled with the device except
in the orientation shown.
[0042] FIG. 10A is a partial side midsectional view of several
sample chambers assembled into a chamber stack, according to one
embodiment of the present invention, exploded to show the cap seals
positioned for insertion.
[0043] FIG. 10B shows the cap seals engaged within the sample
chambers of FIG. 10A.
[0044] FIG. 11A is a front (or equivalently, back) orthogonal view
of an exemplary immobilized buffer disk (IBD) of the present
invention having an ovoid (pseudoelliptical) shape. FIG. 11B gives
an exemplary formula for designing a disk with such shape.
[0045] FIGS. 12A-12F are scanned images of 2D gels--obtained by
immobilized pH gradient (IPG) isoelectric focusing, using either pH
3-10 strips, pH 4-7 strips, or pH 6-10 strips, as indicated
immediately below each gel image, followed by
SDS-PAGE--demonstrating the effects of prefractionating a rat liver
tissue lysate into five separate fractions using a device,
components, and methods of the present invention. FIG. 12A is
obtained with unfractionated lysate. Each of FIGS. 12B-12F is
obtained using a fraction from a different one of the device sample
chambers; the pH range of the device sample chamber is shown in
large type below the IPG strip pH range.
[0046] FIGS. 13A-13E are scanned images of 2D gels. FIG. 13A is
obtained from the pI 4.6-5.4 lysate fraction using a pH 4-7 IPG
strip, FIG. 138 from the pI 4.6-5.4 lysate fraction using a pH
4.5-5.5 narrow range IPG strip, with FIG. 13C showing an
enlargement of the indicated region of the gel shown in FIG. 13B.
FIG. 13D is obtained from unfractionated rat liver lysate using a
pH 4.5-5.5 IPG strip, with FIG. 13E showing an enlargement of the
indicated region of the gel shown in FIG. 13D.
[0047] FIGS. 14A and 14B show an exemplary embodiment of a cathode
plug of the present invention. FIG. 14A is a top perspective
schematic view of the plug, without the electrode wire,
particularly indicating the outlet for traversal of the wire from
the plug interior to its exterior, and the circumferential detent
near the plug bottom, around which the electrode wire wraps. FIG.
14B shows the plug of FIG. 14A with the electrode wire passing from
the electrode, through the plug interior, through the outlet, and
around the circumferential detent.
DETAILED DESCRIPTION OF THE INVENTION
[0048] In a first aspect, the invention provides a device, and
components thereof, for solution phase electrophoretic separation
of analytes within a sample.
[0049] The device comprises an anode, a cathode, a chamber stack
disposed between the anode and cathode, and chamber stacking
means.
[0050] As will be more fully described herein below, the chamber
stack comprises a plurality of detachable sample chambers aligned
along the electrical axis between the anode and cathode. The lumens
of the coaxially aligned sample chambers are collectively capable
of defining an electrically-conductive fluid column through the
chamber stack.
[0051] The chamber stack further comprises a plurality of
junctional partitions, each of the partitions positioned at a
different one of the junctions between adjacent sample chambers.
The partitions prevent bulk fluid flow between the chambers that
are separated by the partitions. The partitions are permeable to
small ions, however, and permeable to at least a plurality of the
analytes in the sample, permitting both ion and at least some
analyte flow therethrough.
[0052] The chamber stacking means is disposed completely external
to the chamber stack. The stacking means facilitates the assembly
of the chamber stack prior to electrophoresis and, during use,
helps maintain the fluid integrity of the chamber stack.
[0053] A schematic depiction of an embodiment of the device of the
present invention is presented in FIG. 1.
[0054] The various components, including lid 1, are viewed from a
perspective above the proximal end of the partially disassembled
device. As used herein, the terms "proximal" and "distal" refer to
the position of a component relative to the anode when the device
of the present invention is assembled and ready for use.
[0055] The embodiment shown in FIG. 1 shows seven sample chambers
2, but the number of sample chambers can be two, three, four, five,
six, seven, eight, nine, ten or more, and still be within the scope
of the present invention.
[0056] FIG. 2A shows several components of the device of FIG. 1 at
greater magnification. The lid is omitted for clarity.
[0057] In the embodiment shown, cathode buffer chamber 8 is
integral with spill trough 7, and anode buffer chamber 9 is
integral with loading tube 4. Such integral manufacture is not
required, however, and either or both of chambers 8 and 9 may be
discrete components; when discrete, either or both of chambers 8
and 9 may be capable of resting within, or being engaged to or
engaged within, spill trough 7, or in other embodiments configured
to rest upon a flat surface.
[0058] In yet other embodiments, the electrical axis may be
vertical during operation. In such embodiments, cathode buffer
chamber 8 may be integral with spill trough 7 and anode buffer
chamber may be integral with loading tube 4, or either or both such
chambers may be discrete components.
[0059] During electrophoresis, such as solution phase isoelectric
focusing, sample chambers 2 are maintained in a coaxial orientation
within loading tube 4. The chambers and other components that are
inserted into loading tube 4 during electrophoresis (described in
more detail below), are referred to collectively as the chamber
stack 5. Chambers 2 are typically sealed during electrophoresis
with cap seals 3. During electrophoresis, chamber stack 5 is held
in place within the loading tube 4 by the screw cap 6, which
applies a circumferentially uniform, proximally-directed, axial
pressure on the chamber stack 5.
[0060] Once the chamber stack 5 is secured in the loading tube 4 by
screw cap 6, the loading tube is lowered into the spill trough 7
and the distal end of screw cap 6 sealably engaged to the cathode
buffer chamber 8.
[0061] FIG. 2B shows the components of FIG. 2A fully assembled.
[0062] The axis of the electric field 12 present during
electrophoresis defines the axis along which the sample chambers
are commonly aligned. The axis is defined by a line linking the
submerged cathode and the submerged anode (further described
below), and is shown by the bold dashed line in FIG. 2B.
[0063] FIG. 3A shows the spill trough 7 and various components of
the cathode region of the device shown in FIG. 1.
[0064] In the embodiment shown, spill trough 7 provides a reservoir
to catch any electrophoresis buffer that might leak, a useful
safety feature, and also provides underlying structural support for
other components of the device. Spill trough 7 is optional,
however, and is omitted in other embodiments. As would be apparent,
in these latter embodiments, cathode buffer chamber 8 is designed
to be a discrete component separate from the spill trough.
[0065] With further reference to FIG. 3A, cathode plug 15 is the
component housing both the cathode electrode 13 and the cathode
wire 14.
[0066] Cathode electrode 13 is the point of electrical contact of
the device with the negative terminal of an external power supply
(not shown). Cathode electrode 13 is in electrical contact with
cathode wire 14. In the embodiment shown, cathode wire 14 extends
from the cathode electrode through the interior of cathode plug
15.
[0067] In other embodiments, cathode wire 14 is routed down the
outside of cathode plug 15. Routing cathode wire 14 through the
interior of cathode plug 15 presents certain advantages, however.
For example, if cathode plug 15 is made of an insulative material,
routing the conductive wire inside the insulative material reduces
or eliminates off-axis line charge, which could cause an
asymmetrical field.
[0068] As shown in FIG. 3B, cathode plug 15 is engaged for use
within cathode port 16, thus bringing cathode wire 14 into contact
with the interior of buffer chamber 8.
[0069] In some embodiments, such as those shown in FIGS. 3A and 3B,
cathode plug 15 is removably engageable with the cathode buffer
chamber. By removably engageable is meant that the user can remove
and replace it in the cathode port without damaging the device.
[0070] In such embodiments, cathode plug 15 is inserted prior to
use into cathode port 16, as shown in FIG. 3B. In one such
embodiment, cathode plug 15 simply rests in the cathode port 16. In
another embodiment, cathode plug 15 snaps into the cathode port 16.
In yet another embodiment, the cathode plug 15 screws into the
cathode port 16.
[0071] In certain removable plug embodiments, cathode plug 15 is
integral with or removably engaged with the lid 1, and is inserted
into cathode port 16 when lid 1 is engaged with cathode tab 17 and
anode tab (further described below).
[0072] In alternative embodiments, cathode plug 15 is permanently
or semi-permanently sealed within the cathode port 16, and may even
be integral therewith.
[0073] The nature of the connection between the cathode plug and
the cathode port can vary but still be within the scope of the
invention.
[0074] The cathode buffer chamber 8 is located at the distal end of
the spill trough 7. The cathode buffer chamber 8 is filled during
operation (i.e., during electrophoresis) with an electrically
conductive cathode buffer which provides electrical connectivity
between the cathode wire 14 and the lumen of chamber stack 5 within
the loading tube 4.
[0075] The cathode buffer chamber 8 optionally further comprises a
cathode buffer inlet 18 (see FIG. 3A) for the introduction (or
removal) of cathode buffer. The cathode buffer inlet 18 may
optionally have a shape distinguishable from the anode buffer inlet
(shown and discussed herein) to ensure the user does not introduce
the wrong buffer into the cathode buffer chamber 8. In the
embodiment shown in FIGS. 3A and 3B, the cathode buffer inlet 18 is
shaped like a "minus sign" (-). Such identifying indicia may, in
addition or in the alternative, be present elsewhere on cathode
buffer chamber 8.
[0076] In certain embodiments, cathode buffer chamber 8 is
transparent, permitting direct visualization of dyes capable of
migrating to the cathode during electrophoresis.
[0077] Cathode buffer chamber 8 may usefully be designed to have a
volume of 10 ml, 15 ml, even 16 ml, 17 ml, 18 ml, 19 ml, or 20 ml
or more.
[0078] Spill trough 7 may also comprise a cathode tab 17 suited to
fit through the cathode tab slot 44 in lid 1 when the device is
fully assembled (see FIGS. 9A and 9B and description below). The
size and/or shape of the cathode tab 17 may optionally be distinct
from the size and/or shape of the anode tab (discussed herein
below) to ensure that the lid, and thus the electrode cables, may
only be attached with the proper polarity. In the embodiment shown
in FIGS. 2A and 2B, cathode tab 17 is narrower (shorter) than anode
tab 23.
[0079] FIG. 4A shows the loading tube and various components of the
anode region of the device shown in FIG. 1 from a distal
perspective, viewed from above.
[0080] Loading tube 4 holds the components of the chamber stack in
coaxial alignment during assembly and operation, as discussed below
with reference to FIG. 5A.
[0081] Anode plug 21 is the component housing both the anode
electrode 19 and the anode wire 20.
[0082] Anode electrode 19 is the point of electrical contact of the
device with the positive terminal of an external power supply (not
shown). Anode electrode 19 is in electrical contact with anode wire
20. In the embodiment shown, anode wire 20 extends from the anode
electrode through the interior of anode plug 21.
[0083] In other embodiments, anode wire 20 is routed down the
outside of anode plug 21. Routing anode wire 20 through the
interior of anode plug 21 presents certain advantages, however. For
example, if anode plug 21 is made of an insulative material,
routing the conductive wire inside the insulative material reduces
or eliminates off-axis line charge, which could cause an
asymmetrical field.
[0084] Anode plug 21 is engaged for use within anode port 22, thus
bringing anode wire 20 into contact with the interior of anode
buffer chamber 9.
[0085] In some embodiments, anode plug 21 is removably engageable
with the anode buffer chamber. By removably engageable is meant
that the user can remove and replace it in the anode port without
damaging the device.
[0086] In such embodiments, anode plug 21 is inserted prior to use
into anode port 22, as shown in FIG. 4B. In one embodiment, anode
plug 21 simply rests in the anode port 22. In another embodiment,
anode plug 21 snaps into the anode port 22. In another embodiment,
the anode plug 21 screws into the anode port 22.
[0087] In certain removable plug embodiments, anode plug 21 is
integral with or removably engaged with the lid 1, and is inserted
into anode port 22 when lid 1 is engaged with cathode tab 17 and
anode tab 23.
[0088] In alternative embodiments, anode plug 21 is permanently or
semi-permanently sealed within the anode port 22, and may even be
integral therewith.
[0089] The nature of the connection between the anode plug and the
anode port can vary but still be within the scope of the
invention.
[0090] Anode buffer chamber 9 may also comprise an anode tab 23
configured to fit through the anode tab slot 42 in lid 1 when the
device is fully assembled. The size and/or shape of the anode tab
23 may optionally be distinct from the size and/or shape of the
cathode tab (discussed above) to ensure that the lid, and thus the
electrode cables, may only be attached with the proper polarity. In
the embodiment shown in FIGS. 4A and 4B, the anode tab 23 is wide
(or long) relative to the cathode tab.
[0091] The anode buffer chamber 9 is filled during operation (i.e.,
during electrophoresis, such as IEF) with an electrically
conductive anode buffer, which provides electrical connectivity
between the anode wire 20 and the lumen of the chamber stack within
the loading tube.
[0092] Anode buffer chamber 9 may usefully be designed to have a
volume of 10 ml, 15 ml, even 16 ml, 17 ml, 18 ml, 19 ml, or 20 ml
or more.
[0093] The anode buffer chamber 9 optionally comprises an anode
buffer inlet 24 for introduction (or removal) of anode buffer. The
anode buffer inlet 24 may optionally have a shape distinguishable
from the cathode buffer inlet 18 (shown and discussed above, FIGS.
3A and 3B) to ensure that the user does not introduce the wrong
buffer into the anode buffer chamber 9. In the embodiment shown in
FIGS. 4A and 4B, the anode buffer inlet 24 is shaped like a "plus
sign" (+).
[0094] In certain embodiments, anode buffer chamber 9 is
transparent, which permits ready visualization of dyes that are
capable of migrating to the anode during electrophoresis.
[0095] FIG. 5A shows the components of the chamber stack, partially
disassembled, from a proximal perspective, viewed from above. FIG.
5B shows the same components from a distal perspective, also viewed
from above.
[0096] When fully assembled, the chamber stack comprises a
plurality of detachable sample chambers aligned along the
electrical axis between the anode and cathode; the lumens of the
coaxially aligned sample are capable collectively of defining an
electrically-conductive fluid column through the chamber stack.
[0097] The chamber stack further comprises a plurality of
junctional partitions, each of the partitions positioned at a
different one of the junctions between the sample chambers. The
partitions prevent bulk fluid flow between partitioned chambers,
but are both ionically conductive and porous to at least a
plurality of the analytes in the sample.
[0098] For use in solution phase isoelectric focusing, the
partitions have additional features; such partitions are referred
to herein as Immobilized Buffer Disks (IBD).
[0099] An IBD is a thin membrane disk containing covalently
attached buffers of defined pH. For solution phase IEF, IBDs
differing in pH are disposed between successive pairs of sample
chambers. The IBD most proximal to the anode is the most acidic,
and the IBD closest to the cathode is the most basic. The other
IBDs are arranged in decreasing order of acidity as they approach
the cathodic end of series of sample chambers. In the embodiment
shown in the accompanying figures, a total of six different IBDs
are used. In one embodiment, the pHs of the six IBDs, from anode to
cathode, are 3.0, 4.6, 5.4, 6.2, 7.0, and 10.0.
[0100] The first six of the seven sample chambers shown in FIG. 5A,
counting from the anode end piece, are shown preassembled. Although
only the seventh chamber is shown disassembled, the junctions
between all the chambers comprise the components shown explicitly
for chamber seven, as discussed below. The various components are
discussed first, by way of example, with respect to sample chamber
seven, with reference to FIGS. 6A and 6B, and then for all
chambers.
[0101] Sample chambers 2 have a through bore, or lumen, that
extends from the proximal face 32, closest to the anode when in the
assembled chamber stack, through the distal face 34, closest to the
cathode when in the assembled chamber stack, as illustrated in
FIGS. 6A and 6B. FIGS. 1, 2A, 5A, 6A and 6C all show the proximal
face of the illustrated sample chambers, whereas FIGS. 5B and 6B
show the distal faces of the respectively illustrated sample
chambers.
[0102] FIG. 6A shows a sealing O-ring 29 positioned near its
operational position at proximal face 32 of the sample chamber 2.
The proximal face 32 comprises seating means for sealing O-ring 29.
In the embodiments shown, the seating means include two concentric
projections 33 from a base level. When assembled, the sealing
O-ring 29 seats around the second, smaller projection, and against
the first of these projections, as illustrated in FIG. 6C.
[0103] In typical embodiments, proximal projections 33 typically
conform substantially in shape to the shape of the internal lumen
of chamber 2. For example, when the lumen is circular, the proximal
projections will also typically be circular.
[0104] Also shown in FIG. 6A is a spacer 30, shown in exploded view
near the distal face 34.
[0105] The distal face 34 of chamber 2 is best illustrated in FIG.
6B. In contrast to FIG. 6A, an IBD 28 is shown instead of a spacer
30. In typical embodiments, spacer 30 and IBD 28 are alternatives,
and only one of the two is seated in the distal recess 36 in the
distal face of sample chambers 2. Spacer 30 will typically have
outer dimensions substantially identical to that of IBD 28, and
substantially identical thickness. In contrast to IBD 28, however,
spacer 30 will have a through bore with shape and dimensions
substantially conformal to the shape and dimensions of the chamber
lumen. The positioning of a spacer between adjacent chambers thus
serves effectively to combine the lumens of the chambers between
which it is positioned into a single sample chamber of increased
volume.
[0106] FIG. 6C shows sealing O-ring 29 assembled on proximal
projections 33 of chamber 2.
[0107] O-ring 29 acts as a face seal between adjacent chambers:
upon assembly, axial compression of the chamber stack compresses
O-ring 29 axially onto the surface of either an IBD 28 or spacer 30
seated within distal recess 36 of the next most proximal chamber in
the stack. The O-ring holds the IBD or spacer in operational
position within distal recess 36, and prevents a free electric path
from becoming established around an IBD partition.
[0108] The same O-ring contemporaneously creates a gland seal
against the walls of the distal recess of the next most proximal
chamber in the stack. This aspect of the seal is not activated by
axial loading, but instead results from the close fit between the
proximal projections 33 and the outer walls of the distal recess 36
which are precisely sized to incorporate the compliant O-ring
between them to form the gland seal. This gland-seal function of
the single O-ring seal prevents leakage of fluid between the
chambers, and conversely prevents external contamination from
entering chambers 2. Hence a single seal accomplishes both face
seal and gland seal functions.
[0109] In other embodiments, an additional O-ring may be positioned
within the distal recess of chamber 2, typically external to (i.e.,
distal to) the IBD or spacer. In yet other embodiments, the O-ring
could be positioned first into the distal recess rather than the
proximal projection as the two adjacent chambers are assembled.
[0110] The structure of the chamber stack as a whole is most easily
understood by reference to the process for its assembly. Although
illustrated here by a method of assembly, no particular order or
method of assembly is required to create a device according to the
present invention.
[0111] Anode end piece 27 is the first (that is, most proximal)
component assembled in the chamber stack (see FIG. 5A). Like
chambers 2, anode end piece 27 has a throughbore, or lumen,
extending from its proximal face through its distal face. The lumen
is typically, in both shape and dimensions, substantially identical
to that of the chambers to be assembled thereto.
[0112] Also like chambers 2, the anode endpiece may include O-ring
seating means on its proximal face. In such case, an O-ring is
applied thereto. In other embodiments, however, no O-ring seating
means are provided, with seal to anode buffer chamber 9 effected,
e.g., with an O-ring integrated into the distal face of anode
buffer chamber 9.
[0113] Anode endpiece 27 typically comprises a distal recess,
analogous to the distal recess on chambers 2.
[0114] A spacer 30, an IBD 28, or an alternative partition is
typically placed in the anode endpiece distal recess.
[0115] In embodiments in which an IBD 28 is used, the first sample
chamber may be bounded on both sides by an IBD, and will therefore
be able to circumscribe a discrete electrophoretic fraction. When a
spacer (with or without a nonconductive semipermeable membrane) is
used, the first sample chamber will be unable to circumscribe a
discrete electrophoretic fraction.
[0116] In other embodiments, an alternative partition may be used.
For example, the partition may be an ion-permeable membrane that is
nonporous to analytes above a selected molecular weight. If the
molecular weight cutoff is small, the partition acts to keep
analytes from traveling into the anode buffer chamber.
[0117] Next, a first sample chamber, with O-ring assembled on the
proximal projections, is added. Alternatively, the O-ring is seated
in the distal recess of anode endpiece 27. In this latter
embodiment, proximally-directed axial pressure later urges the
O-ring onto the proximal projections of the first sample
chamber.
[0118] The first sample chamber may include an IBD or spacer in its
distal recess at the time of its assembly to the anode end piece.
Alternatively, the chamber may be assembled to the anode end piece
without a spacer or IBD, which is thereafter placed in the distal
recess.
[0119] A second sample chamber, with O-ring seated on its proximal
projections, is then added, and the process is repeated until the
desired number of sample chambers has been added. In an
alternative, the O-ring is seated in the distal recess of the first
chamber, and the second chamber then assembled thereto;
proximally-directed axial pressure then urges the O-ring onto the
proximal projections of the next most distal chamber.
[0120] The embodiment illustrated in FIGS. 5A and 5B includes seven
total sample chambers, although the invention also contemplates the
use of fewer or more chambers.
[0121] A cathode end piece 26 (FIG. 5C and in greater detail in
FIG. 8) is the last (that is, most distal) component assembled in
the chamber stack (see FIG. 5A). Like chambers 2, cathode end piece
26 has a throughbore, or lumen, extending from its proximal face
through its distal face. The lumen is typically shaped and
dimensioned substantially identically to that of the chambers to be
assembled thereto.
[0122] Also like chambers 2, the cathode endpiece typically
includes O-ring seating means on its proximal face.
[0123] Cathode endpiece 26 typically includes a distal recess, into
which a spacer, an IBD, or an alternative partition, and
additionally or alternatively, an O-ring, are seated.
[0124] In the embodiments described above, junctions between sample
chambers comprise either an IBD or a spacer. A sample chamber that
is not bounded at both proximal and distal faces by an IBD when
assembled, but rather by spacers, is referred to as a "blank"
chamber.
[0125] Zero, one, two, three, four or more blank chambers may be
used depending on any given application of choice. For a device
with "n" sample chambers, if there are fewer than "n+1" different
IBD filters available, blank sample chambers are required to
properly fill the loading tube. Alternatively, a user of the device
may have access to n+1 IBDs but may chose not to use all of them,
in which case blank sample chambers are also used. Users of the
device may opt to use fewer than all available IBDs if the amount
or number of proteins to be separated is limited, or if he or she
is only interested in proteins in a specific range of pI, or for
other reasons. The number of sample chambers in the IEF device, the
number of blank sample chambers, the number of IBDs and their pHs
can vary but still be within the scope of the present
invention.
[0126] The loading tube 4 can be used to facilitate assembly of the
chamber stack by holding successive components as they are added,
or the chamber stack can be assembled without aid of the loading
tube and subsequently inserted into it. FIG. 5C shows the chamber
stack loaded in the loading tube.
[0127] A removable endcap may be used to constrain the chamber
stack within the loading tube.
[0128] In useful embodiments, the endcap is capable of applying a
proximally directed axial pressure to the chamber stack, urging the
chambers and endpieces together, thus facilitating their sealing
engagement to one another and to the anode and cathode buffer
chambers. In particularly useful embodiments, the endcap is capable
of applying a circumferentially uniform, proximally directed, axial
pressure. FIG. 5D shows end cap 6 engaged to stacking tube 4,
constrained a chamber stack within the tube.
[0129] In one series of embodiments, both loading tube 4 and end
cap 6 are at least partially threaded, and the thread of the end
cap is capable of engaging the thread of the stacking tube (such
embodiments of the end cap are called "screw caps" herein). In
particularly useful embodiments, the stacking tube thread is
external to the stacking tube, and the screw cap thread is internal
to the screw cap.
[0130] The end cap has a lumen, thus permitting fluid communication
between the cathode buffer chamber and the interior of the chamber
stack (i.e., with lumens of the cathode end piece and the
distal-most sample chamber).
[0131] In typical embodiments of the device and components of the
present invention, sample chambers 2 include fill ports 35 (see
FIGS. 6A and 6B), into which cap seals 3 may be sealingly
engaged.
[0132] In embodiments in which the fully engaged cap seals 3 are
not flush with the outer surface of the sample chambers, loading
tube 4 may usefully include an axially oriented channel 25 (see,
e.g., FIGS. 4A and 4B) to accommodate the projection of engaged cap
seals 3 from the body of sample chambers 2. In such embodiments,
cap seals 3 may usefully be inserted into fill ports 35 of sample
chambers 2 prior to inserting the chambers into the loading tube 4:
the necessary engagement of the projecting cap seals within (or
through) the channel during loading ensures that the fill ports of
all of the chambers are aligned within the assembled chamber
stack.
[0133] Usefully, channel 25 of loading tube 4 is positioned at the
top side of loading tube 4, aligning the fill ports of chambers 2
of the chamber stack upwards (see, e.g., FIGS. 5C and 5D). This
facilitates both fluid addition and removal while the chamber stack
is fully assembled.
[0134] In addition, chambers 2 may in some embodiments usefully be
self-indexing--that is, configured automatically to align their
fill ports.
[0135] For example, the proximal projections and the distal
recesses may usefully lack rotational symmetry. In such
embodiments, once the proximal projection of a second chamber is
properly mated within the distal recess of a first chamber, the two
cannot rotate with respect to one another, ensuring that their fill
ports are aligned. Such self-indexing cannot be achieved by
chambers that mate by screwing onto one another, since differences
in membrane thickness and tightening torque cause the final
orientation of any fill port to be arbitrary.
[0136] In certain embodiments, the chamber lumen may advantageously
conform substantially to such rotationally nonsymmetric shape.
[0137] Conforming the shape of the lumen to the shape of the
proximal projections and distal recess minimizes the area of the
IBD outside the "active" field area, which minimizes the amount of
analyte that migrates into the IBD outside the "active" field area
and is lost to the fractionation process.
[0138] Conforming the shape of the lumen to the shape of the
proximal projections also makes diffusive losses from the active to
inactive areas of the IBD uniform around the IBD circumference, by
making uniform the distribution of inactive IBD areas.
[0139] Hence, in certain useful embodiments, the chamber lumen
itself follows the shape of the seal, offset inwardly and uniformly
from the internal edge of the distal recess.
[0140] The lumen of the sample chamber may also advantageously be
nonsymmetric across the horizontal plane (i.e., when the device is
horizontal in use).
[0141] In some embodiments, for example, the chamber lumen may
usefully have a more acute radius of curvature at the bottom than
at the top: as liquid sample is removed after fractionation, this
geometry facilitates the pooling of the remaining fluid at the
bottom of the chamber, thus facilitating the complete withdrawal of
the sample after electrophoresis. In other embodiments, the more
acute radius of curvature is at the top: this facilitates
expression of air from the chamber as the chamber is capped before
electrophoresis.
[0142] FIGS. 7A and 7B present orthogonal midsectional views of an
embodiment of a sample chamber of the present invention, viewed
along line A-A of FIG. 6C. FIG. 7B illustrates the operational
engagement of cap seal 3 within fill port 35. In the embodiment
shown, the chamber lumen is a rotationally nonsymmetric ovoid
(pseudoellipse) (further described below).
[0143] FIGS. 7C and 7D present orthogonal midsectional views of
another embodiment, in which the fill port 35, and the
corresponding portion of the cap seal 3, is tapered. The cap seal
fits in the fill port tightly enough to prevent sample evaporation
but is readily removable by hand. FIG. 7E shows an orthogonal view
of the cap seal of FIG. 7D, from the bottom. The concentric circles
define the cross-sectional areas at three planes within the cap
seal, as described in more detail below.
[0144] FIG. 9A illustrates the lid 1 for the device shown in FIG.
1. Lid 1 protects the user from electrical shock and, when spill
tray 7 includes distinguishable anode and cathode tabs, helps to
ensure proper electrical connection polarity.
[0145] Lid 1 optionally comprises an anode cable 41 and a cathode
cable 43 that connect the anode electrode 19 and the cathode
electrode 13 to their respective outlets on an external power
supply (not shown) when the lid is operationally seated on the
device, as shown in FIG. 9B. The electrode cables can be
permanently attached to lid 1 or removable therefrom.
[0146] In one embodiment, the electrode cables 41 and 43 are
permanently or semi-permanently attached to the lid such that they
are not intended to be removed by the user. In such embodiments,
the electrode cables are optionally color coded to facilitate
connection with proper polarity. In some of these embodiments, the
electrodes are positioned in the lid so that they become submerged
in the anode and cathode buffer chambers when the lid is placed
onto the apparatus, without the intermediation, respectively, of
anode and cathode plugs.
[0147] In other embodiments, the electrode cables 41 and 43 are
detachable from the lid 1. In such embodiments, holes in the lid
above the anode and cathode electrodes allow the electrodes to
protrude, or cables to enter the device, to effect electrical
connection. In another embodiment (not shown), prominent markings
on the lid, e.g. color coded markings or "plus" and "minus" signs,
may be placed on the lid at or near the anode and cathode holes to
clearly indicate to the user which power cord to attach to which
electrode.
[0148] In certain embodiments, lid 1 also comprises an anode tab
slot 42 and a cathode tab slot 44, which optionally differ in size
and/or shape. This size and/or shape difference makes it possible
to attach the lid in only one orientation with respect to the
electrodes, i.e. with a defined electrical polarity.
[0149] The lid also enhances safety by preventing human contact
with the electrode or buffers during operation, since the interior
of the device can only be accessed with the lid removed, which
necessarily disconnects the device from the external power
supply.
[0150] Lid 1 may optionally, but advantageously, be transparent, to
permit visualization of the loading tube, chamber stack, and other
components during electrophoresis.
[0151] To perform solution phase IEF using a device of the present
invention, the chamber stack is first assembled using a plurality
of IBDs, each having a different, fixed, pH. A protein sample of
interest is introduced into one or more sample chambers through
their respective fill ports, and sealed without trapped air using,
for example, a tapered cap seal and a tapered fill port. Any sample
chambers not filled with protein are filled with sample diluent,
which will typically have a low salt concentration. Appropriate
anode and cathode buffers are introduced through the respective
anode and cathode buffer chamber inlets, and electrode plugs are
screwed into the electrode ports. The lid is then put on the
device, and the electrodes connected to the appropriate terminals
on an external power supply.
[0152] An electric potential is applied to the device until each
protein reaches the chamber bounded by IBDs that bracket its
pI.
[0153] The electrical potential is generally at least about 50 V,
100 V, 150 V, or 200V to about 1000 V, 1500 V, even as high as
2,000 V-3,000, with values between 50 V and 3000 V useable. The
potential may be changed within this range during
electrophoresis.
[0154] Power is turned off, the lid is taken off and samples are
removed from the sample chambers and stored as IEF fractions
defined by the IBDs bounding each Chamber.
[0155] The device and methods of the present invention do not
require sample mixing or recirculation during fractionation, or
means therefor. In other embodiments, however, the entire device,
such as that shown in FIG. 9B, is placed on a rocking or rotary
platform during electrophoresis. The gentle, externally applied,
motion of the sample chambers mixes samples in order, for example,
to prevent precipitation of proteins at the surface of the IBD
membranes and/or electrodecantation.
[0156] External agitation of the device of the present invention is
preferable, for example, to recirculation of sample through long
tubing and through peristaltic pumps, as in several prior devices.
Such recirculation exposes samples to greatly increased surface
area, with consequent losses of surface-adherent components, e.g.
proteins. Recirculation also requires that the volume of sample be
increased substantially beyond the volume of the sample chamber.
Surface losses and dilution are particularly disadvantageous with
small amounts of protein.
[0157] External agitation is also superior to internal agitation
using stir bars in each sample chamber. Unlike stir bar devices,
the device of the current invention, coupled with external
agitation, does not require introduction of a foreign part into
each sample, and does not involve a series of fragile moving
parts.
[0158] The device of the present invention enables solution phase
IEF fractionation of relatively small volumes of sample, e.g. about
0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, even 1.0, 1.5, 2.0, and 3.0 ml,
with intermediate values permissible, such as 0.61, 0.62, 0.63,
0.64, 0.65, 0.66, 0.67, 0.68, and 0.69 as exemplary volumes.
Relatively small scale protein preparations can be used in such
devices, making it possible to perform solution IEF as a
prefractionation step on a number of samples prior to analysis on
2D gels.
[0159] The device of the present invention can concentrate protein
samples as well as fractionate them.
[0160] In contrast, many of the prior devices capable of solution
phase IEF are most suited to solution IEF of preparative scale
protein samples, rather than analytical scale samples.
Further Advantages of Component Embodiments
Electrode Plugs
[0161] Cathode plug 15, illustrated in FIGS. 3A and 3B, and anode
plug 21, illustrated in FIGS. 4A and 4B, are collectively referred
to herein as electrode plugs.
[0162] As described above, the electrode plugs can usefully be
removable from their respective ports for ease of cleaning, repair
or replacement. In particularly useful embodiments, the electrode
plugs are identical to or otherwise interchangeable with one
another: this permits the plugs to be used at either end, and
permits a single spare electrode plug to serve as a replacement for
either a cathode plug or an anode plug that is lost or damaged.
[0163] Typically, the electrode plugs according to the present
invention are substantially cylindrical.
[0164] FIGS. 14A-14B show an exemplary embodiment of a cathode plug
(equivalently, anode plug) in which the electrode wire
advantageously passes through the inside of an insulative plug.
[0165] FIG. 14A is a perspective schematic view from above of such
an embodiment, without the electrode wire, particularly indicating
the outlet 45 for traversal of the wire from the plug interior to
its exterior, and an optional circumferential detent 46 near the
plug bottom, around which the electrode wire wraps. FIG. 14B shows
the plug of FIG. 14A with the electrode wire passing from
electrode, through the insulative plug interior, out through the
outlet, and around the circumferential detent.
[0166] Circumferential positioning of the electrode wire renders
the circle of cathode wire co-planar with the circle of anode wire
during use. This, in turn, ensures that the shortest distance
between the anode and cathode remains the same regardless of the
final rotational position of either plug within its respective
port. This eliminates variation in the field strength imposed by
inconsistencies during assembly or re-assembly.
[0167] Recessing the wire within a circumferential detent on the
electrode plug protects the electrode wires from being bent or
broken during handling, particularly cleaning. Such damage is a
common problem with prior devices in which electrode wires are not
fully protected. Consequently, electrode wires in the electrode
plugs of the present invention need not be as thick as electrode
wires in other devices, with consequent cost savings. These
advantages apply particularly to electrode wires comprising
platinum.
[0168] Loading Tube
[0169] The loading tube holds the anode end piece, sample chambers,
IBDs and optionally spacers, and cathode end piece in the proper
coaxial alignment, both during assembly and in operation. As
described in detail above, the entire chamber stack can be
assembled by stepwise addition of new components, using the loading
tube to support those components already assembled. Once assembled,
the components are necessarily axially aligned along the electric
field gradient.
[0170] In certain embodiments, loading tube 4 is transparent,
permitting readily visualization of the chamber stack within the
tube, facilitating correct assembly of the chamber stack and
monitoring for problems within the stack, such as leakage, during
electrophoresis.
[0171] Certain embodiments of the loading tube include axial
channel 25, usefully positioned through the top surface of the
loading tube. The channel accommodates cap seals that do not make a
flush engagement with the sample chambers, and positioned at the
top surface of the loading tube, usefully aligns the chamber fill
ports upwardly.
[0172] Upward positioning of the fill ports allows loading and
unloading of the liquid solutions with a minimum of spillage. In
addition, upward positioning of the fill ports allows gas bubbles
to float into and be captured a region that is capable of serving
as a bubble trap (see below). As would be readily understood, if
the fill port were too far from vertical, i.e., on the side or at
the bottom of the sample chamber then liquid would spill out when
it was opened, and gases formed during the electrophoresis would
rise up to the top of the sample chamber and be trapped there
rather than in the bubble trap.
[0173] In certain embodiments, the anode end piece, which may lack
a fill port, includes a protrusion. The protrusion of the anode end
piece, like the projections of the sample chamber cap seals, is
accommodated by channel 25 of loading tube 4. The anode end piece
protrusion is useful in disengaging all components of the chamber
stack out of the loading tube during disassembly of the device.
[0174] End Cap
[0175] End cap 6 provides a proximally directed axial force that
ensures a tight seal between components of the chamber stack during
assembly and use. In some embodiments, as described above, end cap
6 is a screw cap.
[0176] Use of an end cap readily permits a circumferentially
uniform axial pressure to be applied to the chamber stack; such
circumferentially uniform pressures are difficult to achieve with
prior devices that require four separate nuts to be tightened onto
independent alignment rails.
[0177] In embodiments in which end cap 6 is a screw cap, engagement
of the sample chamber cap seals within the loading tube channel
prevents the sample chambers from rotating relative to one another
and/or to the loading tube as the screw cap is tightened. Rotation
of chambers and/or end pieces of the chamber stack may also be
constrained by use of self-indexing, rotationally nonsymmetric
proximal projections and distal recesses.
[0178] Tapered Cap Seal and Fill Port
[0179] FIG. 10A is a partial side midsectional view of several
sample chambers assembled into a chamber stack, according to one
embodiment of the present invention, exploded to show the cap seals
positioned for insertion. FIG. 10B shows the cap seals engaged
within the sample chambers of FIG. 10A.
[0180] The fill ports provide a variety of advantages.
[0181] First, the aligned fill ports in the assembled chamber stack
allows for the loading of sample and/or buffers into the sample
chambers after the apparatus is fully assembled, rather than
serially during a manual assembly process, as is the case with
other devices.
[0182] Second, the ports allow the fractionated samples to be
extracted, e.g. with a pipette, directly from each chamber
immediately after electrophoresis rather than, as is the case with
other devices, from individual chambers as the device is
dissembled. This reduces the risks of contamination and loss of
sample due to spillage. Moreover, the contents of each chamber can
be removed in any order rather than in the order of disassembly.
For example, it may be desirable to extract the electrophoresis
sample from a particular chamber as quickly as possibly rather than
having to wait for other chambers to be removed first.
[0183] Third, the filling port creates a "bubble trap" that allows
gas bubbles that form during electrophoresis to rise and collect in
a region that is outside of the electric field. This is desirable
because electric current only conducts through the solution and not
through the air/gas bubbles, and the analytes are present only in
solution. Not only can air bubbles thus distort the electric field,
they have been known to form in other electrophoretic systems and
expand to the point where the gas actually occludes the fluid path
to such a degree that it blocks the conduction path and therefore
the entire electrophoretic process. By allowing bubbles to collect
outside of the electric field, the fill ports help to maintain the
field constant in cross-section and therefore uniform in density
and electromotive force (EMF) across the entire IBD surface
throughout the electrophoretic run.
[0184] In some embodiments, as illustrated in FIGS. 7C and 7D, the
walls defining fill port 35 are usefully designed so that air or
other gas or supernatant fluid is expressed, rather than trapped,
as cap seal 3 is inserted into fill port 35 to seal the sample
chamber. In the embodiments shown in FIGS. 7C and 7D, the tapered
cap seal does not fully block the fill port until the cap seal is
fully inserted, giving any air in the fill port the opportunity to
escape prior to closure.
[0185] In such embodiments, the volume of trapped air that is
compressed upon final engagement of the cap seal within the fill
port is reduced, reducing the pressure within the sample chamber
and keeping the pressure below that which destructively displaces
the gel from the IBD, or causes a fluidic leak. This feature also
limits or prevents contamination of the fluid due to airborne
substances in the sample chamber.
[0186] Alternatively, or in addition, the tapered cap seal may be
used to displace a supernatant fluid (other than air) overlaying
the sample in the sample chamber and fill port. For example, in
circumstances where the sample volume is less than that required to
completely fill the sample chamber, it may be desirable to add a
supernatant fluid to displace any air that would otherwise remain
in the sample chamber. Such fluid may be added to fill not only the
sample chamber but also a portion of the fill port, to ensure that
absolutely no air remains. When the tapered cap seal is inserted
into the fill port it will displace any excess supernatant fluid
from the fill port (causing it to overflow out from the top) before
finally sealing the sample chamber. The combination of a
supernatant fluid to displace air, and the tapered cap seal to
allow escape of excess supernatant fluid, facilitates sealing of
sample chambers without unwanted air bubbles.
[0187] Any one of a number of supernatant fluids may be used to
displace air from sample chambers. The only requirements for the
fluid are that it not be miscible with the sample (which will
typically be in aqueous solution), that it be less dense than the
sample, and that its components not interact in undesirable ways
with the sample. For example, mineral oil or 1-butanol may usefully
be employed as an air displacing supernatant fluid.
[0188] With reference to FIG. 7D, the dimensions of the tapered
region 37 of a tapered cap seal may be expressed as the ratio of
the cross sectional area of the cap seal at the top 39 of the
conical region divided by the cross sectional area at the bottom 40
of the conical section. FIG. 7E presents an orthogonal view from
the bottom of the tapered cap seal. The smallest, inner circle in
FIG. 7E represents the cross sectional area at the bottom of the
conical section, and the mid-sized circle represents the cross
sectional area at the top of the conical section. The outermost
circle is the cross sectional area of the knob.
[0189] The taper can be expressed as the ratio of the areas of the
mid-sized and inner circles, or by the percentage by which the
cross sectional area of the mid-size circle exceeds the cross
sectional area of the inner circle. In the embodiment represented
in FIG. 7E, the area of the mid-sized circle is approximately 4.4
times the area of the inner circle. For this embodiment, the cross
sectional area at the top of the conical region of the cap seal
exceeds the cross sectional area at the bottom of the conical
region by 340%. In other embodiments of the present invention (not
shown), the cross sectional area at the top of the conical region
of the cap seal exceeds the cross sectional area at the bottom of
the conical region only slightly, or by 20%, 50%, 100%, 200%, 400%
or more. The precise value of the taper may vary but still be
within the scope of the present invention.
[0190] Immobilized Buffer Disks (IBDs)
[0191] In another aspect, the invention provides immobilized buffer
disks (IBDs; synonymously, "disks"); in certain embodiments, the
IBDs are particularly adapted for use in the devices of the present
invention.
[0192] As used herein, the term "disk" does not intend that the IBD
necessarily present a circular surface as viewed along the
electrical axis of the device; the shape of the IBD will typically
conform to the shape of the sample chamber lumen, which as further
described herein may advantageously lack rotational symmetry.
[0193] The IBDs of the present invention, when positioned as
partitions between adjacent sample chambers in a chamber stack, are
capable of interrupting bulk fluid flow through the chamber stack,
but are nonetheless permeable to ions and at least a plurality of
analytes desired to be analyzed. For use in solution phase
isoelectric focusing, the disks have a fixed pH.
[0194] The IBDs comprise a porous support and a gel. In typical
embodiments, the gel at least partially fills voids within the
support.
[0195] In general, the support material should provide voids for
gel inclusion and lack facial charges. The support can be
constructed, for example, from glass fiber microfilter materials,
such as Whatman GF/A, GF/B, GF/C, and GF/D filter material (Whatman
Inc., Clifton, N.J., USA). In other embodiments, the support can be
constructed of polyethylene, such as flash-spun polyethylene
(Tyvek.RTM., E.I. DuPont de Nemours, DE, USA), fritted polyethylene
(Porex Corp., Fairburn, Ga., USA), sintered polyethylene, or bonded
spun polyester fibers. In yet other embodiments, the support can
include cellulose filters, cotton nonwoven fabrics, and nylon tulle
fabric.
[0196] The supports of the present invention are typically thin,
ranging in thickness from about 0.1 mm, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 to about 1.5 mm, preferably
from about 0.4 to about 1.0 mm, most preferably from about 0.6 to
about 0.8 mm. In one embodiment, the supports of the present
invention have a width of about 0.65 mm, typically from about
0.64-0.68 mm.
[0197] Typically, the completed IBD is at most insubstantially
thicker than the support, and the IBD thus typically ranges in
thickness from about 0.1 mm, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.1, 1.2, 1.3, 1.4 to about 1.5 mm, preferably from about
0.4 to about 1.0 mm, most preferably from about 0.6 to about 0.8
mm. In one embodiment, the IBDs of the present invention have a
width of about 0.65 mm, typically from about 0.64-0.68 mm.
[0198] The support is impregnated (i.e., its void volume
substantially filled) with the gel material (acrylamide, agarose,
etc.) of choice.
[0199] In embodiments in which the gel is a polyacrylamide gel,
some formulations previously used in the art result in gel that
tends to "ooze out" of the disks. In order to prepare disks having
a minimal thickness that can nonetheless meet the structural
requirements, the exemplary formulations set forth in Table 1,
below, may be used.
TABLE-US-00001 TABLE 1 EXEMPLARY IBD FORMULATIONS % Crosslinker
Ratio of Acrylamide as w/w % of acrylamide to Formula (w/v)
acrylamide crosslinker w/w I 5 4 25 II 10 4 25 III 5 8 12.5 IV 5 10
10 V 6 8 12.5 VI 6 10 17 VII 7 3 33 VIII 7 4 25 IX 7 5 20 X 7 6 17
XI 7 8 12.5 XII 7 9 11 XIII 7 10 10
[0200] In an IBD of the present invention, the w/v percentage of
the total acrylamide concentration in the final gel (% T) can be as
low as 4%, although % T is typically higher, such as 5%, 6%, 7%,
and may be as great as 8%, 9%, even 10%, or more, with nonintegral
values permissible within the acceptable range. The percent w/w of
crosslinker to total acrylamide (% C) may be as low as 4%, and may
be as high as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% even as
high as 15%, with nonintegral values permissible within this
range.
[0201] When used for solution phase isoelectric focusing, the IBDs
will have a fixed pH, and the gel of the IBD will typically further
comprise at least one species of copolymerized pH-conferring
monomer.
[0202] In one series of embodiments, the copolymerized
pH-conferring monomer is an acrylamido buffer monomer. As is known
in the art, acrylamido buffers are non-amphoteric weak acids and
bases having a vinyl moiety for incorporation into the gel
matrix.
[0203] Acrylamido buffer monomers useful in the IBDs of the present
invention are known in the art. A number of commercially available
acrylamido buffers (Amersham Biosciences, Piscataway, N.J., USA,
and Sigma-Aldrich, St. Louis, Mo., USA) include:
2-acrylamido-2-methylpropane sulfonic acid; 2-acrylamidoglycolic
acid; N-acryloylglycine; 4-acrylamidobutyric acid;
2-morpholinoethylacrylamide; 3-morpholinopropylacrylamide;
N,N-dimethylaminoethylacrylamide;
N,N-dimethylaminoethylpropylacrylamide; and
N,N-diethylaminopropylacrylamide.
[0204] In some embodiments, the gel will include a copolymer of
acrylamide, N,N'-methylene-bis-acrylamide, and at least one species
of acrylamido buffer monomer.
[0205] In other embodiments, the copolymerized pH-conferring
monomer is a dicarboxylic acid.
[0206] The dicarboxylic acid may usefully have the formula:
##STR00001##
in which R is selected from the group consisting of: --H, --OH,
--CH.sub.2OH, --CO.sub.2H, --NHR', --OCH.sub.3, and --NR'R'', --Cl,
--F, --I, and wherein R' and R'' are each independently selected
from the group consisting of --CH.sub.3, --CH.sub.2CH.sub.3,
--CH.sub.2OH, --CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.3.
[0207] Use of dicarboxylic acids as copolymerized pH-conferring
monomers presents significant advantages. The advantages are
particularly evident for IBDs having pH fixed in the range of about
5.0 to about 6.0 with itaconic acid as the pH-conferring
monomer.
[0208] Previously, disks and strips (such as IPG strips) in the pH
range of about 5.0 to 6.0 have required high percentages of
acrylamido buffers, for example as much as 10% v/v of gel, in order
to achieve adequate buffering strength. This is because the two
acrylamido buffers that bracket the desired pH have pKa values of
about 4.6 (4-acrylamidobutyric acid) and about 6.2
(2-morpholinoethylacrylamide). The buffering capacity of a compound
falls off logarithmically from the buffer's pKa. Thus, a fairly
large amount of a base is required to titrate the pH of
4-acrylamidobutyric acid from 4.6 to 5.4, and a fairly large amount
of an acid is required to titrate the pH of
2-morpholinoethylacrylamide from 6.2 to 5.4.
[0209] High percentages of acrylamido buffer disadvantageously lead
to non-uniform electrical fields, physical instability of the gel,
and increase cost. They also tend to disadvantageously increase the
hydrophobicity of the gels, leading to protein retention. High
percentages of acids and bases additionally cause, or at least
increase the amount or rate of, breakdown of polyacrylamide,
decreasing shelf life.
[0210] In contrast, embodiments of the IBDs of the present
invention that include a gel that comprises a copolymerized
dicarboxylic acid monomer are capable of having a desirably low
"buffer ratio". By "buffer ratio" is meant the ratio (mol/mol) of
buffer (e.g., pH conferring monomer plus titrant) to gel. In
certain embodiments of the IBDs of the present invention, the
buffer ratio may be less than about 15%, less than about 10%, less
than about 5%, preferably less than about 1%, and most preferably
less than about 0.1%.
[0211] By using less pH conferring monomer and titrant,
dicarboxylic acid-containing gels of the present invention
typically have a longer shelf-life compared to other gels and
systems that do not utilize itaconic acid or other dicarboxylic
acids.
[0212] In some embodiments, the IBD is stable when stored at
4.degree. C. or at ambient temperature for at least 2 weeks to
about 3, 4, 5, 6, 7, or 8 weeks, more preferably from about 1 month
to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, most
preferably from about 1 year to about 2 or 3 years.
[0213] In certain embodiments, the dicarboxylic acid monomer may be
copolymerized with an acrylamido buffer monomer, typically further
copolymerized with acrylamide and an acrylamide crosslinker, such
as N,N'-methylene-bis-acrylamide.
[0214] Although here described as a copolymerized component of IBDs
of the present invention, dicarboxylic acids can be used as gel
monomers in a variety of electrophoretic devices and methods, and
are thus not limited to the compositions and methods disclosed
herein.
[0215] The IBDs can usefully further include visible indicia, which
may appear on either or both sides of the disk.
[0216] The indicia can be incorporated using, for example, the
methods and compositions disclosed in co-pending and commonly owned
U.S. Pat. No. 6,521,111 B1, published U.S. patent application US
2003/0038030 A1, and published PCT patent application WO 01/77655
A1. Such indicia include, without limitation, any one or more of
the pH of the IBD, the manufacturer, the date of manufacture, the
lot number, a trademark, and any other distinctive or useful mark.
Exemplary trademarks include, without limitation, ZOOM.RTM.,
Invitrogen', NOVEX.RTM. and the Invitrogen design trademark (U.S.
registration #75912326).
[0217] Alternatively, the indicia can be printed directly on the
disk using, e.g., ink jet printing approaches.
[0218] After polymerization, IBDs may be washed; washing may
usefully reduce contaminants, such as unpolymerized monomers,
buffer, or catalyst.
[0219] The IBDs may be washed, for example, in a low ionic strength
solution buffered near neutrality. The wash solution can
conveniently be based on the low ionic strength buffers described
in U.S. Pat. Nos. 5,578,180, 5,922,185, 6,059,948, 6,096,182,
6,143,154, 6,162,338, the disclosures of which are incorporated
herein by reference in their entirety.
[0220] For example, the wash solution can usefully comprise BisTris
((2-hydroxyethyl)iminotris(hydroxymethyl)methane), Tricine,
glycerol and/or sorbitol, EDTA, sodium azide, and SB-14
(3-(N,N-dimethylmyristylammonio)propanesulfonate), titrated to a
neutral pH.
[0221] In addition or in the alternative, the IBDs can be washed
with one or more reducing agents, such as those included in the
running buffers described, e.g., in U.S. Pat. No. 5,578,180, the
disclosure of which is incorporated herein by reference in its
entirety. The reducing agent can, e.g., be sodium bisulfite.
[0222] IBDs that are particularly adapted for use in the device of
the present invention will typically conform in shape to the lumen
of the sample chamber, albeit with dimensions sufficiently larger
than those of the chamber lumen as to permit the IBD to seat within
the distal recess without passing into the lumen itself. In
preferred embodiments, the amount by which the IBD exceeds the
chamber lumen in size will be uniform around the entirety of the
IBD circumference.
[0223] As described above, the lumen of the sample chambers of the
present invention may usefully lack rotational symmetry. And as
further described above, the lumen of the sample chambers of the
present invention may advantageously lack symmetry across the
horizontal plane. Accordingly, in certain embodiments, the IBDs of
the present invention lack rotational symmetry, and in other
embodiments additionally lack symmetry across the horizontal
plane.
[0224] A variety of shapes comprising circular arcs of varying
radii and separation distances can be designed that meet such
criteria, including a variety of elliptical and pseudo-elliptical
shapes, such as an ovoid shape.
[0225] In one series of embodiments, the IBD of the present
invention is ovoid, as illustrated in FIG. 11A: this
pseudoelliptical shape advantageously lacks rotational symmetry and
is nonsymmetric across the horizontal plane.
[0226] In one series of embodiments, the ovoid shape may be defined
by the following general formulae, in which the arcs and angles are
identified in FIG. 11B:
"Base"
Arc:x.sup.2+y.sup.2=R.sub.B.sup.2|.sub..angle.1.sup..angle.2
"Side 1"
Arc:(x-x').sup.2+y.sup.2=R.sub.S.sup.2|.sub..angle.2.sup..angle-
.3
"Point"
Arc:x.sup.2+[y-(-y')]=.sup.2=R.sub.p.sup.2|.sub..angle.3.sup..an-
gle.4
"Side 2"
Arc:[x-(-x')].sup.2+y.sup.2=R.sub.S.sup.2|.sub..angle.4.sup..an-
gle.1 (2) [0227] Where R.sub.S1=R.sub.S2 [0228] and |-x'|=x' [0229]
and R.sub.P<R.sub.B<R.sub.S
[0230] In a particular one of these embodiments, for example,
R.sub.S1=R.sub.S2=11.89 mm; R.sub.P=5.26 mm; R.sub.B=6.37 mm;
<1=0.degree.; <2=180.degree.; <3=213.76.degree.;
<4=326.24.degree.; X'=|-X'|=5.52 mm; and (0, -Y')=(0, -3.68 mm).
This shape and size are suitable for use with a chamber having a
lumenal volume of about 650-750 .mu.l.
[0231] Variations from this exemplary shape are within the scope of
this invention. For example, the separation (y) of centerlines
between the "base" and "point" arcs (dimension Y' in the figures)
may be as low as about 3.0 mm, and as high as about 25.4 mm. That
is, the separation can be any value, including by way of
non-limiting example, about 3, about 4, about 5, about 6, about 7,
about 8, about 9, about 10, about 11, about 12, about 13, about 14,
about 15, about 16, about 17, about 18, about 19, about 20, about
21, about 22, about 23, about 24 or about 25 mm. Fractions and
sub-fractions of the preceding separation values can also be used.
For example, in the case of separations between about 3 to about 4
mm, the separation can be about 3.0, about 3.1, about 3.2, about
3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about
3.9 or about 4.0 mm. As a further example, in the case of
separations between about 3.0 to about 3.1 mm, the separation can
be about 3.00, about 3.01, about 3.02, about 3.03, about 3.04,
about 3.05, about 3.06, about 3.07, about 3.08, about 3.09 or about
3.10 mm.
[0232] Variations from the exemplary size are also within the scope
of this invention.
[0233] For example, the size can be scaled up or down at any factor
suitable for any chamber lumenal volume. By way of non-limiting
example, for volumes between 1,500 and 500 .mu.l, a disk designed
for a 750 .mu.l chamber lumenal volume could be scaled up or down
by a factor of 5.times. up to 0.3.times., respectively.
[0234] Additional desirable dimensional and shape criteria
optionally include: (1) minimization of the size of the disk; (2)
minimization of the area of the disk outside of the chamber lumen;
(3) provision of a full and adequate seal between adjacent
chambers; (4) maintenance of uniformity of an electrical field that
mobilizes molecules through the disk; and (5) maintenance of a
specific orientation between adjacent seals and surfaces.
[0235] Kits
[0236] In another aspect, the invention provides kits for
performing solution phase isoelectric focusing the device and
components of the present invention.
[0237] In one series of embodiments, the kit provides components
that can be assembled into the device of the present invention. The
kit components may be sufficient to assemble a complete device,
optionally with spare parts, or may instead include only a subset
of device components.
[0238] The kit may, for example, include one or more of a spill
trough with integral cathode buffer chamber, a loading tube with
integral anode buffer chamber, lid with electrodes, and chamber
stack components. The chamber stack components may include O-rings,
sample chambers, sample chamber fill port cap seals, spacers and/or
IBDs, cathode end piece, and anode end piece. The kit may also
include one or more of end screw cap, and electrode plugs.
[0239] In another series of embodiments, the kit provides only
disposable items, such as spacers and/or IBDs.
[0240] For example, the kits may include only IBDs, for example a
plurality of IBDs each having the same fixed pH, such as pH 3.0,
4.6, 5.4, 6.2, 7.0, and 10.0. Each of the plurality of disks may be
separately packaged, or the disks may be physically segregated
within a common package.
[0241] For example, in one embodiment, a plurality of IBDs, such as
2, 3, 4, even 5, 6, 7, 8, 9, or 10 or more IBDs, each having the
same fixed pH, are physically segregated from one another within a
single blister pack or strip, such as those described in U.S. Pat.
No. 4,691,820.
[0242] A blister pack, strip or package typically consists of two
pieces: a base and a cover. The base is an injection-molded plastic
that typically has a bowl-shaped, or rectangular-shaped, recess for
receiving and holding an IBD. The cover is a laminate material that
typically consists of a laminate of an aluminum foil and
polypropylene. The sealing of the cover layer to the base portion,
or its flange, can be carried out by the action of heat or
ultrasound, or by means of some other suitable bonding process.
Once sealed, a series of individual sealed pockets ("blisters") is
formed, each one of which comprises an individual IBD.
[0243] Typically, each blister also comprises a hydrating and/or
buffering solution to prevent drying of the IBD and to maintain the
IBD ready for use. The solution is conveniently a low ionic
strength solution buffered near neutrality, typically further
comprising one or more agents to maintain the suppleness of the
support and one or more preservatives, such as sodium azide and/or
a chelating agent, such as EDTA. The solution can conveniently be
based on the low ionic strength buffers described in U.S. Pat. Nos.
5,578,180, 5,922,185, 6,059,948, 6,096,182, 6,143,154, 6,162,338,
the disclosures of which are incorporated herein by reference in
their entirety.
[0244] For example, the solution can usefully comprise BisTris
((2-hydroxyethyl)iminotris(hydroxymethyl)methane), Tricine,
glycerol and/or sorbitol, EDTA, sodium azide, and SB-14
(3-(N,N-dimethylmyristylammonio)propanesulfonate), titrated to a
neutral pH.
[0245] Typically, the amount of solution in a given blister is
between 0.8 to 5 ml, with most between 1 and 3 ml, typically about
1 ml.
[0246] In another embodiment, each "blister" of a single blister
pack or strip comprises a single IBD, each of the IBDs in the pack
or strip having a different fixed pH, so that one strip contains
all of the requisite IBDs for operation of the device. For example,
a blister pack can contain a series of IBDs having pH values of
3.0, 4.6, 5.4, 6.2, 7.0 and 10.0.
[0247] In yet other embodiments, the kits may include a plurality
of such common pH IBDs, with the plurality including IBDs of a
plurality of pHs, permitting multiple operations of the device of
the present invention.
[0248] In other embodiments, the kits may include, either
separately or in conjunction with any of the kits above-described,
any one or more reagents useful in solution phase isoelectric
focusing, such as carrier ampholytes, anode buffer (e.g., as a
50.times. concentrate), and cathode buffer (e.g., for pH 3-10, at
10.times. concentrate). The kits may also include one or more
reagents for solubilizing and/or denaturing proteins, such as urea,
thiourea, and CHAPS
(3-[(cholamidopropyl)dimethylammonio]-propanesulfonate).
[0249] The kits of the present invention may further comprise one
or more sets of instruction, one or more protein standards, and/or
one or more control samples.
[0250] Yet other kits may commonly package a plurality of IBDs,
with various fixed pHs, with one or more immobilized pH gradient
(IPG) strips having pH range suitable for further analysis of
fractions bracketed by the included IBD pHs.
[0251] Methods
[0252] The device and immobilized buffer disks of the present
invention can readily be used for solution phase isoelectric
focusing (IEF), particularly for solution phase isoelectric
focusing prefractionation of samples prior to further analysis,
such as by 2D PAGE.
[0253] In a typical embodiment of the methods of the present
invention, protein samples are prepared in sample buffer; the
sample chambers and appropriate IBDs are assembled within the
loading tube; the samples are loaded into the sample chambers; the
sample chambers are capped with cap seals; anode buffer is added to
the anode reservoir and cathode buffer is added to the cathode
buffer chamber; fractionation is performed; cap seals are removed
and sample fractions retrieved; and the fractions then used for
downstream analytical applications.
[0254] As would be understood, the steps as listed above, and their
order, are exemplary.
[0255] In a first step of this exemplary method, protein samples
are prepared in sample buffer. Preparation of Samples for
Isoelectric Focusing is known in the art. See, e.g., Rabilloud,
Proteome Research: Two Dimensional Gel Electrophoresis and
Identification Tools, Springer Verlag (2000) (ISBN: 3540657924) and
Rabilloud, Methods Mol. Biol. 112:9-19 (1999), the disclosures of
which are incorporated herein by reference in their entireties.
[0256] As is well known, the major objectives of sample preparation
are to completely solubilize the proteins, denature the proteins,
reduce disulfide bonds, prevent protein modification, and maintain
the proteins in solution during solution phase IEF. Accordingly,
the sample buffer typically contains: urea, for denaturation and
solubilization, and/or thiourea; detergent, such as non-ionic or
zwitterionic detergents, for solubilization, such as CHAPS, NP-40,
CAPSO, and sulfobetaines; DTT or DTE (dithioerythritol), as a
reducing agent; and ampholytes, which help solubilize the proteins
and maintain the pH gradient. Ampholytes are typically used at
concentrations of about 0.2-2% (v/v); higher concentrations require
longer focusing times.
[0257] Optionally, but preferably, the sample proteins can be
reduced and alkylated by treating with DTT followed by alkylation
in the presence of N,N-dimethylacrylamide (DMA). Also optionally,
particulate material can be removed by high-speed centrifugation to
reduce the chance of clogging the IBDs.
[0258] In embodiments of the device of the present invention in
which sample chambers have volumes of about 700 .mu.l, samples are
typically then diluted to about 0.6 mg protein/ml.
[0259] In the next step, the chambers are assembled as described
herein above.
[0260] In one exemplary assembly method, O-rings are placed on the
proximal projections and cap seals within the fill ports of each of
7 exemplary sample chambers. The loading tube is held vertically
and the anode end piece inserted therein. A first (most proximal)
chamber is then inserted into the loading tube with its cap seal
projecting through the loading tube channel. A pH 3.0 IBD is placed
in the first chamber's distal recess. A second chamber is then
inserted into the loading tube, and a pH 4.6 IBD placed in its
distal recess. The process is repeated with IBDs having pH 5.4,
6.2, 7.0 and 10.0. A final (7.sup.th) sample chamber is inserted,
followed by the cathode end piece. The end screw is screwed onto
the loading tube to effect sealing engagement among the sample
chambers within the loading tube.
[0261] As is described in greater detail above, the number of
chambers, and the order of IBDs therebetween, is not limited to
this exemplary embodiment.
[0262] For example, with 7 chambers, the device may be assembled
with fewer than 6 IBDs, using spacers in lieu of one or more
IBDs.
[0263] For example, for fractionating in the pH 4-5 range, the
following exemplary chamber stack order may be used (from proximal
to distal): anode end piece, chamber, pH 3.0 IBD, chamber, spacer,
chamber, pH 4.6 IBD, chamber, pH 5.4 IBD, chamber, spacer, chamber,
pH 10.0 IBD, chamber, cathode end piece.
[0264] For fractionating in the pH 5-6 range, the following
exemplary chamber stack order may be used (from proximal to
distal): anode end piece, chamber, pH 3.0 IBD, chamber, spacer,
chamber, pH 5.4 IBD, chamber, pH 6.2 IBD, chamber, spacer, chamber
pH 10.0 IBD, chamber, cathode end piece.
[0265] For fractionating in the pH 5-7 range, the following
exemplary chamber stack order may be used (from proximal to
distal): anode end piece, chamber, pH 3.0 IBD, chamber, spacer,
chamber, pH 5.4 IBD, chamber, pH 7.0 IBD, chamber, spacer, chamber,
pH 10.0 IBD, chamber, cathode end piece.
[0266] For fractionating in the pH 3-4 range, the following
exemplary chamber stack order may be used (from proximal to
distal): anode end piece, chamber, spacer, chamber, pH 3.0 IBD,
chamber, pH 4.6 IBD, chamber, spacer, chamber, spacer, chamber, pH
10.0 IBD, chamber, cathode end piece.
[0267] Next, the loading tube is inserted into the spill trough so
that the end screw sealingly engages the cathode buffer
chamber.
[0268] Anode buffer and cathode buffer chambers are then filled
with respective buffers.
[0269] Next, the samples are loaded into the sample chambers: cap
seals are removed, and sample added to each nonblank chamber (i.e.,
chamber partitioned on both sides by an IBD). In one exemplary
embodiment, 670 .mu.l is added to each nonblank chamber. Cap seals
are reinserted into the chamber fill ports.
[0270] The lid is then engaged to the spill trough, and the
electrodes attached to a power supply.
[0271] Exemplary electrical parameters are 100 V for 20 minutes,
200 V for 80 minutes, and 600 V for 80 minutes. If the power supply
has a current and power limiting capability, the current limit may
usefully be set at 2 mA and the power limit at 2 W.
[0272] If current is flowing through the system, bromophenol blue
included in the sample migrates towards the anode reservoir,
usefully coloring it yellow as a visual check.
[0273] Following electrophoresis, the power supply is turned off,
the lid removed, the cap seals removed and sample fractions
retrieved. The fractions may usefully be removed using, e.g., a 1
ml pipette tip on a pipettor. The fractions may usefully be
transferred to separate microcentrifuge tubes. To recover all of
the fraction, the chamber may be washed with a wash buffer (e.g.,
sample buffer without any inhibitors).
[0274] The fractions may then be used for downstream analytical
applications.
[0275] For example, after suitable dilution and/or desalting, the
fractions may be subjected to one dimensional electrophoresis using
SDS-PAGE, or 2D liquid chromatography/mass spectrometry (or 2D
LC/MS/MS) analysis.
[0276] Alternatively, the fractions may be applied directly to
immobilized pH gradient (IPG) strips for 2D PAGE analysis.
Typically, neither buffer exchange nor further sample processing is
required prior to IPG IEF, since the fractionated sample is in the
same buffer required for first dimension IEF using IPG strips.
[0277] In one approach, fractions are applied to IPG strips which
have pH range about 0.1 pH unit wider than the nominal pH range of
the solution phase IEF fraction.
[0278] The device and methods of the present invention permit
fractionation of complex samples by solution phase isoelectric
focusing. By so doing, the device and methods of the present
invention allow the loading of increased amounts of protein in
downstream applications, such as 2D-PAGE, reduce sample complexity,
result in high resolution and identification of low abundance
proteins, increase the dynamic range of detection by increasing the
concentration of protein species, and reduce
precipitation/aggregation artifacts of samples at high protein
loads during 2D gel electrophoresis.
[0279] Additional Applications
[0280] Although features and aspects of the device of the present
invention are illustrated above in embodiments of solution IEF
devices, one of skill in the art would recognize that many aspects
described would be advantageous in the field of electrophoresis
generally.
[0281] For example, the use of a loading tube sealed at the end
with an end cap, such as a screw cap, that provides a
circumferentially uniform, axially-directed pressure, would also be
useful in sealing chambers in an electroelution device, or in a
tube gel electrophoresis device.
[0282] As another example, the anode and cathode plugs described
above would be useful in any electrophoresis device. The ease of
cleaning and replacement, the sturdiness, and the ability to use
very thin fragile wire would be advantageous in all forms of
electrophoresis.
[0283] As yet a further example, one of skill in the art would
recognize that the tapered fill port and cap seal arrangement
described above would be generally useful in any device in which a
chamber is desirably to be sealed without trapping air. Any device
in which air bubbles disrupt an electric field, such as
electroelution chambers, free solution electrophoresis devices, and
others would benefit from a sealing mechanism that helps exclude
air.
[0284] The following examples are offered by way of illustration,
not by way of limitation.
Example 1
Fractionation of Rat Liver Lysate
[0285] Rat liver tissue is lysed by sonication at a final
concentration of 5% (w/v) in 7M urea, 2M thiourea, 4% CHAPS
(collectively, "UTC") and protease inhibitors. After reduction,
alkylation, centrifugation, and determination of the protein
concentration of the supernatant fraction, samples are diluted to
0.6 mg/ml protein in UTC containing 1% ZOOM.RTM. ampholytes, pH
3-10 (Invitrogen Corp., Carlsbad, Calif., USA), 20 mM DTT, and a
trace of bromophenol blue dye.
[0286] An aliquot of 3.35 ml is distributed equally into five
central sample chambers of seven total chambers, each with capacity
of about 670 .mu.l, designed and assembled according to the device
of the present invention. The five chambers are partitioned from
one another by IBDs having pH 3.0, 4.6, 5.4, 6.2, 7.0, and
10.0.
[0287] After fractionation for 3 hours, the resulting fractions are
collected and a 155 .mu.l aliquot of each fraction is loaded onto a
separate ZOOM.RTM. IPG strips (Invitrogen Corp., Carlsbad, Calif.,
USA). As a control, an aliquot of 155 .mu.l of unfractionated
sample (92 .mu.g unfractionated rat liver lysate proteins) is
loaded directly onto a ZOOM.RTM. 3-10 NL Strip (Invitrogen Corp.,
Carlsbad, Calif., USA).
[0288] The ZOOM.RTM. Strips are allowed to rehydrate with the
applied samples overnight, and then focused in a ZOOM.RTM.
IPGRunner.TM. System (Invitrogen Corp., Carlsbad, Calif., USA). The
focused ZOOM strips are then applied to NuPAGE.RTM. Novex 4-12%
Bis-Tris ZOOM.RTM. gels. The resulting 2DE gels are stained with
SimplyBlue.TM. SafeStain (Invitrogen Corp., Carlsbad, Calif., USA)
and scanned.
[0289] FIGS. 12A-12F are scanned images of the resulting 2D
gels.
[0290] The pH range of the immobilized pH gradient (IPG) strip is
shown immediately beneath each gel image.
[0291] FIG. 12A is obtained with unfractionated lysate. Each of
FIGS. 12B-12F is obtained using a fraction from a different one of
the device sample chambers; the pH range of the device sample
chamber is shown in large type below the IPG strip pH range.
[0292] The results show that the device of the present invention
can efficiently separate a complex proteome, such as rat liver
tissue, into five well defined fractions based on pH. The
fractionation reduces the sample's complexity while increasing the
concentration of the fractionated proteins.
Example 2
Improvement in Detection of Low Abundance Proteins
[0293] Rat liver lysate is prepared and fractionated in a device of
the present invention, essentially according to Example 1.
[0294] Separate 155 .mu.l aliquots of the pH 4.6-5.4 fraction are
loaded respectively on a pH 4.5-5.5 narrow range ZOOM.RTM. IPG
strip (Invitrogen Corp., Carlsbad, Calif., USA) and a pH 4-7
ZOOM.RTM. IPG strip (Invitrogen Corp., Carlsbad, Calif., USA) and
allowed to rehydrate overnight. The applied proteins are then
focused using the ZOOM.RTM. IPGRunner.TM. System (Invitrogen Corp.,
Carlsbad, Calif., USA). The focused ZOOM.RTM. Strips are separately
applied to NuPAGE.RTM. Novex 4-12% Bis-Tris ZOOM gels. The
resulting 2DE gels are stained with SimplyBlue.TM. SafeStain and
scanned.
[0295] Unfractionated rat liver lysate is analogously applied to a
pH 4.5-5.5 narrow range ZOOM.RTM.IPG strip (Invitrogen Corp.,
Carlsbad, Calif., USA) as a control.
[0296] FIG. 13A shows the solution phase fraction run on a pH 4-7
IPG strip, demonstrating that prefractionation in the device of the
present invention yields a fraction with clearly defined pI
range.
[0297] FIG. 13B is an equivalent solution phase pH 4.6-5.4 fraction
run on a pH 4.5-5.5 narrow range narrow range IPG strip. FIG. 13C
shows an enlargement of the indicated region of the gel shown in
FIG. 13B. By comparison, FIG. 13D is obtained from unfractionated
rat liver lysate using a pH 4.5-5.5 IPG strip, with FIG. 13E
showing an enlargement of the indicated region of the gel shown in
FIG. 13D.
[0298] Comparison of FIGS. 13C and 13E demonstrate that
prefractionation using the device of the present invention improves
the ability to detect low abundance proteins.
[0299] All patents, patent publications, and other published
references mentioned herein are hereby incorporated by reference in
their entirety as if each had been individually and specifically
incorporated by reference herein.
[0300] Examples are intended to illustrate the invention and do not
by their details limit the scope of the claims of the invention.
While preferred illustrative embodiments of the present invention
are described, it will be apparent to one skilled in the art that
various changes and modifications may be made therein without
departing from the invention, and it is intended in the appended
claims to cover all such deviations and modifications that fall
within the true spirit and scope of the invention.
* * * * *