U.S. patent application number 11/614785 was filed with the patent office on 2007-07-05 for compositions and methods for improving resolution of biomolecules separated on polyacrylamide gels.
This patent application is currently assigned to INVITROGEN CORPORATION. Invention is credited to Thomas A. Beardslee, Timothy V. Updyke.
Application Number | 20070151853 11/614785 |
Document ID | / |
Family ID | 38218323 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070151853 |
Kind Code |
A1 |
Beardslee; Thomas A. ; et
al. |
July 5, 2007 |
Compositions and Methods for Improving Resolution of Biomolecules
Separated on Polyacrylamide Gels
Abstract
Gels, such as polyacrylamide gels, are provided that include
linear polyacrylamide in the stacking gel. Native gels that include
linear polyacrylamide in the stacker can be used to separate
biomolecular complexes, such as protein complexes. Gel cassettes in
which the gap width between front and back plates does not vary by
more than 5% at the upper edge of the cassette are also provided.
The gel cassettes can be used for electrophoretic separation of
proteins and protein complexes on native gels, such as native gels
that include linear polyacrylamide in the stacker. The native gels
can have multiple wells for electrophoresing at least one sample
and/or at least one molecular weight standard.
Inventors: |
Beardslee; Thomas A.;
(Carlsbad, CA) ; Updyke; Timothy V.; (Temecula,
CA) |
Correspondence
Address: |
INVITROGEN CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
INVITROGEN CORPORATION
1600 Faraday Avenue
Carlsbad
CA
92008
|
Family ID: |
38218323 |
Appl. No.: |
11/614785 |
Filed: |
December 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60754328 |
Dec 29, 2005 |
|
|
|
60761342 |
Jan 24, 2006 |
|
|
|
Current U.S.
Class: |
204/456 ;
204/606 |
Current CPC
Class: |
C07K 1/26 20130101; C08J
3/075 20130101; C08J 2333/26 20130101; C08K 5/0008 20130101; G01N
27/44756 20130101; C08L 33/26 20130101; G01N 27/44747 20130101;
C08L 33/26 20130101; C08K 5/0008 20130101 |
Class at
Publication: |
204/456 ;
204/606 |
International
Class: |
C07K 1/26 20060101
C07K001/26; G01N 27/00 20060101 G01N027/00 |
Claims
1. A polyacrylamide gel for the separation of biomolecules,
comprising a stacking gel and a separating gel, wherein the
stacking gel comprises linear polyacrylamide.
2. The polyacrylamide gel of claim 1, wherein the acrylamide
concentration of the stacking gel is between 2% and 6%.
3. The polyacrylamide gel of claim 2, wherein the acrylamide
concentration of the stacking gel is between 2.5% and 5%.
4. The polyacrylamide gel of claim 2, wherein the linear acrylamide
concentration of the stacking gel is from 0.005% to 1%.
5. The polyacrylamide gel of claim 4, wherein the linear acrylamide
concentration of the stacking gel is from 0.01% to 0.5%.
6. The polyacrylamide gel of claim 5, wherein the linear acrylamide
concentration of the stacking gel is from 0.02% to 0.1%.
7. The polyacrylamide gel of claim 1, wherein the separating gel
does not comprise linear acrylamide.
8. The polyacrylamide gel of claim 1, wherein the polyacrylamide
gel is a denaturing gel.
9. The polyacrylamide gel of claim 8, wherein the polyacrylamide
gel comprises SDS.
10. The polyacrylamide gel of claim 1, wherein the polyacrylamide
gel is a nondenaturing gel.
11. The polyacrylamide gel of claim 10, wherein the nondenaturing
gel is a gradient gel.
12. The polyacrylamide gel of claim 10, wherein the non-denaturing
gel is a Blue Native Gel.
13. A method of separating biomolecules on an electrophoresis gel,
comprising: applying one or more samples comprising one or more
biomolecules to an acrylamide gel comprising a stacking gel portion
and a separating gel portion, wherein the stacking gel portion
comprises linear polyacrylamide; and electrophoretically separating
one or more biomolecules on the gel.
14. The method of claim 13, wherein the separating gel portion does
not comprise linear acrylamide.
15. The method of claim 13, wherein the electrophoresis gel is a
denaturing gel.
16. The method of claim 13, wherein the electrophoresis gel is a
nondenaturing gel.
17. The method of claim 16, wherein the nondenaturing gel is a Blue
Native gel.
18. The method of claim 16, wherein the nondenaturing gel is a
gradient gel.
19. The method of claim 13, further comprising applying one or more
molecular weight marker sets to the electrophoresis gel.
20. The method of claim 19, further comprising estimating or
calculating the molecular weight of one or more biomolecules or
biomolecular complexes on the electrophoresis gel.
21-41. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority to U.S.
Provisional Application 60/754,328, entitled "Compositions and
Methods for Improving Resolution of Biomolecules Separated on
Polyacrylamide Gels", filed Dec. 29, 2005; and U.S. Provisional
Application 60/761,342, entitled "Compositions and Methods for
Improving Resolution of Biomolecules Separated on Polyacrylamide
Gels", filed Jan. 24, 2006; each of which is herein incorporated by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to electrophoresis methods and
apparatus for minimizing skirting effects in an electrophoretic
gel.
[0004] 2. Background Information
[0005] Gel electrophoresis remains a fundamental technique of
biochemistry, molecular biology, and cell biology for its
usefulness in the separation, characterization, and identification
of biomolecules and molecular complexes. Optimal use of gel
electrophoresis requires separation of biomolecules with high
resolution. One problem confronted by biochemists that employ gel
electrophoresis is "skirting", in which molecules of a sample
loaded on the gel migrate between the gel and a gel plate, rather
than through the gel itself. This leads to the appearance shadow
bands that migrate more quickly than the main bands of the
biomolecule or complex that migrate within the gel. This creates
ambiguity when images of the gel are analyzed, as it is difficult
to know whether such bands are artifacts or the result of a low
abundance biomolecule or complex.
SUMMARY OF THE INVENTION
[0006] Provided herein are electrophoresis gels, cassettes and
methods for reducing the skirting effect present in gel
electrophoresis. In illustrative embodiments, the electrophoresis
gels, cassettes and methods provided herein can be used to reduce
the skirting effect present in non-denaturing gel electrophoresis.
Also provided herein are electrophoresis gels, cassettes and
methods used to reduce the skirting effect present in denaturing
gel electrophoresis
[0007] In one aspect provided herein are electrophoresis gels for
the separation of biomolecules wherein the electrophoresis gels
have a stacking gel and a separating gel, and wherein the stacking
gel includes linear polyacrylamide. In an embodiment of such
electrophoresis gels, the electrophoresis gels are polyacrylamide
gels, wherein the stacking gel and a separating gel are
polyacrylamide gels, and wherein the stacking gel includes linear
polyacrylamide. In further or alternative embodiments the
acrylamide concentration of the stacking gel is between about 2%
and about 6%, while in further or alternative embodiments the
acrylamide concentration of the stacking gel is between about 2.5%
and about 5%. In further or alternative embodiments, the linear
acrylamide concentration of the stacking gel is from about 0.005%
to about 1%, while in further or alternative embodiments, the
linear acrylamide concentration of the stacking gel is from about
0.01% to about 0.5%. In still further or alternative embodiments,
the linear acrylamide concentration of the stacking gel is from
about 0.02% to about 0.1%. In other embodiments the separating gel
does not comprise linear acrylamide.
[0008] In other embodiments of this aspect, the electrophoresis gel
is a denaturing gel, while in further or alternative embodiments
the electrophoresis gel is a polyacrylamide gel that is a
denaturing gel. In further or alternative embodiments, such
denaturing gels include sodium dodecyl sulfate (SDS).
[0009] In other embodiments of this aspect, the electrophoresis gel
is a non-denaturing gel, while in further or alternative
embodiments the electrophoresis gel is a polyacrylamide gel that is
a non-denaturing gel. In further or alternative embodiments, such
non-denaturing gels are gradient gels, while in certain embodiments
such non-denaturing gels are Blue Native Gels.
[0010] Another aspect provided herein are methods for separating
biomolecules on an electrophoresis gel, wherein such methods
include applying one or more samples comprising one or more
biomolecules to an electrophoresis gel that includes a stacking gel
portion and a separating gel portion, wherein the stacking gel
portion comprises linear polyacrylamide; and then
electrophoretically separating the one or more biomolecules on the
electrophoresis gel. In an embodiment of this aspect the separating
gel does not comprise linear acrylamide.
[0011] In further or alternative embodiments of this aspect, the
electrophoresis gel is a denaturing gel, while in further or
alternative embodiments the electrophoresis gel is a polyacrylamide
gel that is a denaturing gel. In further or alternative
embodiments, such denaturing gels include sodium dodecyl sulfate
(SDS).
[0012] In other embodiments of this aspect, the electrophoresis gel
is a non-denaturing gel, while in further or alternative
embodiments the electrophoresis gel is a polyacrylamide gel that is
a non-denaturing gel. In further or alternative embodiments, such
non-denaturing gels are gradient gels, while in certain embodiments
such non-denaturing gels are Blue Native Gels.
[0013] In other embodiments of this aspect, the methods also
include applying one or more molecular weight marker sets to the
electrophoresis gel, and in further or alternative embodiments,
such methods also include estimating or calculating the molecular
weight of one or more biomolecules or biomolecular complexes
electrophoreses on the electrophoresis gel.
[0014] Another aspect provided herein are gel cassettes for
performing gel electrophoresis, wherein the cassette has a
consistent gap width across its cross section. In certain
embodiments of this aspect such cassettes have a consistent gap
width across their upper edge. In certain embodiments of this
aspect such cassettes have a consistent gap width across their
upper edge in the range from 0.1 millimeters to 5 millimeters. In
further or alternative embodiments, the gap width of such cassettes
varies by less than 5%, while in other embodiments the variation in
the gap width of such cassettes is 2% or less. In further or
alternative embodiment, such cassettes contain polyacrylamide gels.
In other embodiments, such cassettes are used for performing
non-denaturing gel electrophoresis, and the gel is a non-denaturing
gel. In certain embodiments, such non-denaturing gels are Blue
Native Gels. In further or alternative embodiment, the gels
contained in such cassettes are gradient gels. In further or
alternative embodiment, such gradient gels are polyacrylamide
gradient gels.
[0015] In further or alternative embodiments of this aspect, the
cassettes are fabricated from plastic, while in further or
alternative embodiments the plastic cassettes are fabricated by
welding together a front plate to a back plate. In further or
alternative embodiments, the welding of the front plate to the back
plate results in a cassette with a consistent gap width.
[0016] Another aspect provided herein are methods for separating
biomolecules on an electrophoresis gel, wherein such methods
include applying one or more samples comprising one or more
biomolecules to an electrophoresis gel contained in a cassette that
has a consistent gap width; and then electrophoretically separating
the one or more biomolecules on the electrophoresis gel.
[0017] In an embodiment of this aspect the cassette has a
consistent gap width across its cross section, while in other
embodiments such cassettes have a consistent gap width across their
upper edge. In certain embodiments of this aspect such cassettes
have a consistent gap width across their upper edge in the range
from 0.1 millimeters to 5 millimeters. In further or alternative
embodiments, the gap width of such cassettes varies by less than
5%, while in other embodiments the variation in the gap width of
such cassettes is 2% or less. In further or alternative embodiment,
such cassettes contain polyacrylamide gels. In other embodiments,
such cassettes are used for performing non-denaturing gel
electrophoresis, and the gel is a non-denaturing gel. In certain
embodiments, such non-denaturing gels are Blue Native Gels. In
further or alternative embodiment, the gels contained in such
cassettes are gradient gels. In further or alternative embodiment,
such gradient gels are polyacrylamide gradient gels. In other
embodiments of this aspect, the electrophoresis gel contained in
such cassettes includes a stacking gel and a separating gel. In
further or alternative embodiments, such stacking gels include
linear acrylamide.
[0018] In further or alternative embodiments of such methods, the
method also includes applying one or more molecular weight marker
sets to the electrophoresis gel. In further or alternative
embodiments, the methods also include estimating or calculating the
molecular weight of one or more biomolecules or biomolecular
complexes electrophoreses on the electrophoresis gel. In further or
alternative embodiments of such methods, the method also includes
staining the gel.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows 3-12% Blue Native gradient gels made without
(top gel) or with (bottom gel) 0.05% linear acrylamide in the
stacking gel.
[0020] FIG. 2 shows a schematic depiction of a cassette (21) sliced
through the middle in which the cassette has a front plate (22) and
a back plate (23) with a gap in between (24).
[0021] FIG. 3 depicts sample proteins/protein complexes and marker
proteins separated on a gel run in a cassette that did not have a
consistent gap width between plates (A) and a gel run in a cassette
that did have a consistent gap width between plates (B).
DETAILED DESCRIPTION OF THE INVENTION
[0022] Disclosed herein are electrophoresis gels, cassettes and
methods used for reducing the skirting effect present in gel
electrophoresis. In illustrative embodiments, the electrophoresis
gels, cassettes and methods provided herein can be used to reduce
the skirting effect present in non-denaturing gel electrophoresis,
while in other embodiments the electrophoresis gels, cassettes and
methods provided herein can be used to reduce the skirting effect
present in denaturing gel electrophoresis.
[0023] Definitions
[0024] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used herein are well known and commonly
employed in the art. Terms of orientation such as "up" and "down",
"top" and "bottom", "above" and "underneath" or "upper" or "lower"
and the like refer to orientation of parts during use of a device.
Where a term is provided in the singular, the inventors also
contemplate the plural of that term. Where there are discrepancies
in terms and definitions used in references that are incorporated
by reference, the terms used in this application shall have the
definitions given herein. As employed throughout the disclosure,
the following terms, unless otherwise indicated, shall be
understood to have the following meanings.
[0025] The term "ambient temperature" as used herein, refers to the
temperature in the range of 20.degree. C. to 25.degree. C.
[0026] As used herein, a biopolymer or biomolecule includes, but is
not limited to, a nucleic acid, a protein, a polysaccharide, a
lipid, and other macromolecules. A nucleic acid includes DNA, RNA,
oligonucleotides, and fragments and analogs thereof. Nucleic acid
sequences may be derived from genomic DNA, RNA, mitochondrial
nucleic acid, chloroplast nucleic acid and other organelles with
separate genetic material.
[0027] The term "chaotropic agent" or "chaotrope", as used herein,
refers to any substance capable of altering the secondary and
tertiary structure of proteins and nucleic acids.
[0028] The term "electrophoresis gel", as used herein, refers to a
gel used for electrophoretic separation of a sample. An
electrophoresis gel can be a separating gel only, or an
electrophoresis gel can include both a stacking gel and a
separating gel.
[0029] The term "Linear polyacrylamide" or "linear acrylamide", as
used herein, refers to linear, non-crosslinked polymers of
acrylamide, and may also be referred to simply as "high molecular
weight acrylamide". Linear acrylamide can be in dry chemical or
liquid form (i.e., as a weight/volume solution) with molecular
weight ranges from 1,000 Daltons to about 6,000,000 Daltons,
corresponding to the lengths of the linear polymers.
[0030] As used herein, proteins are complex, three-dimensional
substances comprising one or more long, folded polypeptide chains.
These chains, in turn, include of small chemical units called amino
acids. All amino acids contain carbon, hydrogen, oxygen, and
nitrogen. Some also contain sulfur. A "peptide" is a compound that
includes two or more amino acids. The amino acids link together in
a line to form a peptide chain. There are 20 different naturally
occurring amino acids involved in the biological production of
peptides, and any number of them can be linked in any order to form
a peptide chain. The naturally occurring amino acids employed in
the biological production of peptides all have the L-configuration.
Synthetic peptides can be prepared employing conventional synthetic
methods, using L-amino acids, D-amino acids or various combinations
of amino acids of the two different configurations. Some peptide
chains contain only a few amino acid units. Short peptide chains,
e.g., having less than ten amino acid units, are sometimes referred
to as "oligopeptides", where the prefix "oligo" signifies "few."
Other peptide chains contain a large number of amino acid units,
e.g., up to 100 or more, and are referred to a "polypeptides",
where the prefix "poly" signifies "many." Still other peptide
chains, containing a fixed number of amino acid units are referred
to using a prefix that signifies the fixed number of units in the
chain, e.g., an octapeptide, where the prefix "octa" signifies
eight. (By convention, a "polypeptide" can be considered as any
peptide chain containing three or more amino acids, whereas an
"oligopeptide" is usually considered as a particular type of
"short" polypeptide chain. Thus, as used herein, it is understood
that any reference to a "polypeptide" also includes an
oligopeptide. Further, any reference to a "peptide" includes
polypeptides, oligopeptides. Each different arrangement of amino
acids forms a different polypeptide chain. In certain non-limiting
examples, the polypeptide includes between 40 and 4000 amino acids,
between 50 and 3000 amino acids, or between 75 and 2000 amino
acids.
[0031] As used herein, a "nucleic acid molecule" refers to the
phosphate ester polymeric form of ribonucleosides (adenosine,
guanosine, uridine or cytidine; "RNA molecules") or
deoxyribonucleosides (deoxyadenosine, deoxyguanosine,
deoxythymidine, or deoxycytidine; "DNA molecules"), or any
phosphoester analogues thereof, such as phosphorothioates and
thioesters, in either single stranded form, or a double-stranded
helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are
possible. The term nucleic acid molecule, and in particular DNA or
RNA molecule, refers only to the primary and secondary structure of
the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes double-stranded DNA found, inter
alia, in linear or circular DNA molecules (e.g., restriction
fragments), plasmids, and chromosomes. In discussing the structure
of particular double-stranded DNA molecules, sequences may be
disclosed herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the non-transcribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA). A "recombinant DNA molecule" is a DNA molecule that has
undergone a molecular biological manipulation. (see Sambrook et al.
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory Press).
[0032] As used herein, non-denaturing gels refer to electrophoresis
gels that do not include denaturing agents (such as, for example,
denaturing detergents, urea, formamide, and other chaotropes).
Non-denaturing (or "native") gels are commonly used in "native" gel
electrophoresis, in which the running buffer and sample buffer also
lack denaturants. These gels can be particularly useful in
investigating molecular interactions, such as for example,
protein:protein interactions, protein-nucleic acid interactions,
etc. and for performing in-gel activity assays.
[0033] The term "polyacrylamide", as used herein, refers to a
mixture of acrylamide monomers and N,N'-methylene bis acrylamide
("bis" or "bisacrylamide"), where the acrylamide and bis have been
crosslinked to form a branched molecular structure.
[0034] The term "sample", as used herein, refers to a mixture of a
plurality of unique molecular species which can be separated using
gel electrophoresis. By way of example only, a sample may be a
mixture of nucleic acids, a mixture of oligonucleotides, a mixture
of DNA, a mixture of RNA, or combinations thereof. In addition, by
way of example only, a sample may be a mixture of amino acids, a
mixture of peptides, a mixture of proteins, or combinations
thereof.
[0035] The term "separating gel" or, alternatively "body of
separating gel" refers to the area of the electrophoresis gel in
which the separation of biomolecules occurs and in which separated
biomolecules of interest are localized after electrophoretic
separation has occurred.
[0036] The term "skirting" or "skirting effect", as used herein,
refers to when a sample is able to migrate between the
electrophoresis gel and the wall of the cassette wall, or the
plastic or glass plate or plates, holding or containing the gel.
The proportion of sample that is able to migrate between the gel
and the cassette wall, or plastic or gel plate(s), migrate faster
than rest of the sample thereby giving the appearance of a shadow
or skirt band.
Electrophoresis Gels, Compositions and Gel Cassettes for Reducing
Skirting Artifacts in Electrophoresis
[0037] The electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein include a body of separating
gel and optionally include a stacking gel. Such separating gels are
used to separate sample components including, but not limited to,
biomolecules, while the stacking gels are used to help focus the
sample components into a narrow band prior to migration into the
separating gel. This focusing allows for enhanced resolution of
closely migrating sample components. The processes by which the
biomolecules separate in the separating gels include, but are not
limited to separation by size, separation by charge, or separation
by a combination of size and charge. Biomolecules separated using
such separating gels are detected by dying, staining or labeling
the biomolecules of interest (before, after, or during
electrophoretic separation) and observing (visualizing) their
position within the separating gel after electrophoretic
separation.
[0038] In certain embodiments of the compositions, gel cassettes
and methods disclosed herein, the dyes, stains, labels or other
indicators are added to the sample prior to loading. In other
embodiments, the dyes, stains, labels or other indicators are added
to the loading well or wells, located in the separating gel or the
stacking gel, prior to addition of the sample to such loading well
or wells. In other embodiments the dyes, stains, labels or other
indicators are added to the loading well or wells after to addition
of the sample to the loading well or loading wells. In certain
embodiments of the compositions, gel cassettes and methods
disclosed herein, the separating gel is exposed to at least one
dye, stain, label or other indicator after the electrophoresis run,
whereby the sample components become associated with such dyes,
stains, labels or other indicators. Alternatively, in certain
embodiments of the compositions, gel cassettes and methods
disclosed herein, the dyes, stains, labels or other indicators are
added to the separation gel whereby they become associated with the
sample components during electrophoretic migration. In still other
embodiments of the compositions, gel cassettes and methods
disclosed herein, the dyes, stains, labels or other indicators are
covalently attached to the sample components. Visualization of the
sample bands in the separating gel is then achieved by illuminating
the separating gel with light of appropriate wavelength(s) to allow
observation of the dyes, stains, labels or other indicators
associated with the sample bands.
[0039] The separating gels of the compositions, gel cassettes and
methods disclosed herein can comprise any material which forms a
gel including, but not limited to, synthetic polymers, natural
polymers and combinations thereof. Examples of such synthetic
polymers include, but are not limited to, linear polyacrylamide,
crosslinked polyacrylamide, and combinations thereof. Examples of
such natural polymers include, but are not limited to,
polysaccharides such as agarose. In certain embodiments of the
compositions, gel cassettes and methods disclosed herein such
separating gels can comprise agarose, polyacrylamide, or
combinations of agarose and polyacrylamide. In certain embodiments
such separating gels can comprise agarose, polyacrylamide, or
combinations of agarose and polyacrylamide. In certain embodiments
the separating gels can comprise linear acrylamide and agarose,
linear acrylamide and polyacrylamide, or linear acrylamide and a
combination of agarose and polyacrylamide.
[0040] The stacking gels of the compositions, gel cassettes and
methods disclosed herein can comprise any material which forms a
gel including, but not limited to, synthetic polymers, natural
polymers and combinations thereof. Examples of such synthetic
polymers include, but are not limited to, linear polyacrylamide,
crosslinked polyacrylamide or combinations thereof. Examples of
such natural polymers include, but are not limited to,
polysaccharides such as agarose. In certain embodiments of the
compositions, gel cassettes and methods disclosed herein such
stacking gels can comprise agarose, polyacrylamide, or combinations
of agarose and polyacrylamide. In certain embodiments the stacking
gels comprise linear acrylamide and agarose, linear acrylamide and
polyacrylamide, or linear acrylamide and a combination of agarose
and polyacrylamide.
[0041] In certain embodiments of the compositions, gel cassettes
and methods described, the electrophoresis gels include a
separating gel and a stacking gel, in which the stacking gel
includes linear polyacrylamide. The inclusion of linear acrylamide
in the stacking gel minimizes or prevents skirting artifacts.
[0042] In certain embodiments of the compositions, gel cassettes
and methods disclosed herein, the separating and stacking gels are
polyacrylamide gels, where the stacking gel also includes linear
polyacrylamide. In certain embodiments of the compositions, gel
cassettes and methods disclosed herein, the separating and stacking
gels are polyacrylamide gels, where the separating gel also
includes linear polyacrylamide. In certain embodiments of the
compositions, gel cassettes and methods disclosed herein, the
separating and stacking gels are polyacrylamide gels, where both
the separating gel and the stacking gel include linear
polyacrylamide.
[0043] The polyacrylamide gels of the compositions, gel cassettes
and methods disclosed herein are made using solutions of
"acrylamide" that are mixtures of monomeric acrylamide and
bisacrylamide. The polymerization of acrylamide and bisacrylamide
using polymerization initiators, and if needed catalysts, to
produce crosslinked polyacrylamide gel. The ratios of monomeric
acrylamide to bisacrylamide used in the mixtures to make the
polyacrylamide gels of the compositions, gel cassettes and methods
disclosed herein range from about 15:1 to about 50:1. By way of
example only, the (monomeric) acrylamide:bisacrylamide ratio in
such polyacrylamide gels can be 15:1, 19:1, 24:1, 29:1, 37.5:1,
40:1, 45:1 and 50:1. In certain embodiments disclosed herein, the
ratios of (monomeric) acrylamide to bisacrylamide for the analysis
of proteins and protein complexes, are in the range from about 19:1
to about 45:1.
[0044] In certain embodiments of the electrophoresis gels used in
the compositions, gel cassettes and methods disclosed herein the
stacking gel comprises polyacrylamide made using the mixtures of
acrylamide and bisacrylamide as described above. In certain
embodiments, the stacking gel is made with lower acrylamide
concentration than that used to make the separating gel. By way of
example only, stacking gels can have (w/v) acrylamide
concentrations ranging from about 2% to about 8%, from about 2.5%
to about 7.5% acrylamide, from about 3% to about 7% acrylamide,
from about 3.5% to about 6.5% acrylamide, from about 4% to about 6%
acrylamide, from about 4.5% to about 5.5% acrylamide, or from about
2.5% to about 6% acrylamide. By way of example only, stacking gels
can have (w/v) acrylamide concentrations ranging from 2% to 8%,
from 2.5% to 7.5% acrylamide, from 3% to 7% acrylamide, from 3.5%
to 6.5% acrylamide, from t 4% to 6% acrylamide, from 4.5% to 5.5%
acrylamide, or from 2.5% to 6% acrylamide.
[0045] In certain embodiments, the separating gels and stacking
gels (individually or together) of the electrophoresis gels used in
the compositions, gel cassettes and methods disclosed herein
include linear polyacrylamide. In other embodiments, the
polyacrylamide separating gels and polyacrylamide stacking gels
(individually or together) of the electrophoresis gels used in the
compositions, gel cassettes and methods disclosed herein include
linear polyacrylamide. The (w/vol) concentrations of the linear
acrylamide included in such gels can range from about 0.005% to
about 1%, 0.005% to about 0.75%, 0.005% to about 0.5%, 0.005% to
about 0.1%, 0.01% to about 1%, 0.01% to about 0.75%, 0.01% to about
0.5%, 0.01% to about 0.1%, 0.02% to about 1%, 0.02% to about 0.75%,
0.02% to about 0.5%, 0.02% to about 0.2%, or 0.02% to about 0.1%.
In illustrative embodiments, the (w/vol) concentrations of the
linear acrylamide included in such gels can range from 0.005% to
1%, 0.005% to 0.75%, 0.005% to 0.5%, 0.005% to 0.1%, 0.01% to 1%,
0.01% to 0.75%, 0.01% to 0.5%, 0.01% to 0.1%, 0.02% to 1%, 0.02% to
0.75%, 0.02% to 0.5%, 0.02% to 0.2%, or 0.02% to 0.1%. In addition,
the molecular weight of the linear acrylamide included in such gels
can range from about 1,000 Daltons to about 6,000,000 Daltons, from
about 1,000 Daltons to about 5,000,000 Daltons, from about 1,000
Daltons to about 2,000,000 Daltons, from about 1,000 Daltons to
about 1,000,000 Daltons, from about 1,000 Daltons to about 750,000
Daltons, from about 1,000 Daltons to about 500,000 Daltons, from
about 1,000 Daltons to about 300,000 Daltons, from about 1,000
Daltons to about 200,000 Daltons, from about 1,000 Daltons to about
100,000 Daltons, from about 1,000 Daltons to about 50,000 Daltons,
from about 1,000 Daltons to about 25,000 Daltons, or from about
1,000 Daltons to about 10,000 Daltons. In addition, the molecular
weight of the linear acrylamide included in such gels can range
from 1,000 Daltons to 6,000,000 Daltons, from 1,000 Daltons to
5,000,000 Daltons, from 1,000 Daltons to 2,000,000 Daltons, from
1,000 Daltons to 1,000,000 Daltons, from 1,000 Daltons to 750,000
Daltons, from 1,000 Daltons to 500,000 Daltons, from 1,000 Daltons
to 300,000 Daltons, from 1,000 Daltons to 200,000 Daltons, from
1,000 Daltons to 100,000 Daltons, from 1,000 Daltons to 50,000
Daltons, from 1,000 Daltons to 25,000 Daltons, or from 1,000
Daltons to 10,000 Daltons. A skilled artisan can test linear
acrylamide of various molecular weight ranges to determine useful
molecular weights for linear polyacrylamide used in stacking gels.
In some embodiments, the molecular weight of linear polyacrylamide
used in the stacking gel of the electrophoresis gels used in the
compositions, gel cassettes and methods disclosed herein is greater
than about 10,000 Daltons. In some embodiments, the molecular
weight of linear polyacrylamide used in the stacking gel of the
electrophoresis gels used in the compositions, gel cassettes and
methods disclosed herein is greater than about 100,000 Daltons. In
some exemplary embodiments, the molecular weight of linear
polyacrylamide used in the stacking gel of the electrophoresis gels
used in the compositions, gel cassettes and methods disclosed
herein is between about 100,000 Daltons and about 1,000,000
Daltons. In some exemplary embodiments, the molecular weight of
linear polyacrylamide used in the stacking gel of the
electrophoresis gels used in the compositions, gel cassettes and
methods disclosed herein is between about 600,000 Daltons and about
1,000,000 Daltons. In some exemplary embodiments, the molecular
weight of linear polyacrylamide used in the stacking gel of the
electrophoresis gels used in the compositions, gel cassettes and
methods disclosed herein is between 600,000 Daltons and 1,000,000
Daltons.
[0046] In certain embodiments, the polyacrylamide separating gels
and polyacrylamide stacking gels that include linear acrylamide, as
disclosed herein, are made by the polymerization of a mixture that
includes at least the following mixture of linear acrylamide,
monomeric acrylamide, bisacrylamide crosslinker, and a
polymerization initiator or initiators. This mixture can optionally
include a catalyst. Polymerization of such mixtures can be
initiated by any suitable means which are well known to those
skilled in the art including, chemical initiation by adding
suitable agents and optional catalysts; photochemical initiation
using a photoinitiator followed by irradiation at a suitable
wavelength; thermal initiation, and combinations thereof.
Polymerization of such mixtures incorporates the linear acrylamide
into the polymerized gel; thereby strengthening the gel. In
addition, the incorporation of linear acrylamide into the stacking
gel reduces or eliminates the skirting effect.
[0047] The chemical initiators that can be used to initiate the
polymerization of such mixtures includes, but are not limited to
ammonium persulfate, ammonium persulfate and
tetramethylethylenediamine (TEMED) mixtures, sodium persulfate,
sodium persulfate and tetramethylethylenediamine (TEMED) mixtures,
potassium persulfate, potassium persulfate and
tetramethylethylenediamine mixtures, peroxides, benzyl peroxide,
dicumyl peroxide, azobis [2-(2-imidazolin-2-yl) propane] HCl
(AZIP), azobis (2-amidinopropane) HCl (AZAP),
4,4'-azo-bis-4-cyanopentanoic acid, azobisisobutyramide;
azobisisobutyramidine.2HCl, 2-2'-azo-bis-2-(methylcarboxy) propane,
2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone, and
2-hydroxy-2-methyl-1-phenyl-1-propanone. The photoinitiators that
can be used to initiate the polymerization of such mixtures
includes, but are not limited to, acetophenones, benzophenones,
multi-ringed quinones, fluoresceins, azobisnitriles, benzoquinones,
xanthophenones, benzoins, xanthones, fluoroenones, anthroquinones,
eosin, erythrosin, nitroxides, ribolflavin, riboflavin
5'-phosphate, and derivatives thereof.
[0048] In certain embodiments, the linear acrylamide is added to a
monomeric acrylamide solution prior to adding bisacrylamide
crosslinker, initiator(s), and optional catalyzing agent(s),
thereby resulting in the mixture of linear acrylamide, monomeric
acrylamide, bisacrylamide crosslinker, polymerization initiator(s)
and optional catalyst(s) used to make polyacrylamide separating
gels and polyacrylamide stacking gels that includes linear
acrylamide as disclosed herein. In certain embodiments, the linear
acrylamide is added to a monomeric acrylamide solution prior to
adding bisacrylamide crosslinker, ammonium persulfate and
tetramethylethylenediamine (TEMED), thereby resulting in the
mixture used to make polyacrylamide separating gels and
polyacrylamide stacking gels that includes linear acrylamide as
disclosed herein.
[0049] In certain embodiments, the linear acrylamide is added to a
solution of monomeric acrylamide solution and bisacrylamide
crosslinker prior to adding initiator(s) and optional catalyzing
agent(s), thereby resulting in the mixture of linear acrylamide,
monomeric acrylamide, bisacrylamide crosslinker, polymerization
initiator(s) and a catalyst (s) used to make polyacrylamide
separating gels and polyacrylamide stacking gels that includes
linear acrylamide as disclosed herein. In certain embodiments, t he
linear acrylamide is added to a solution of monomeric acrylamide
solution and bisacrylamide crosslinker prior to adding ammonium
persulfate and tetramethylethylenediamine (TEMED), thereby
resulting in the mixture used to make polyacrylamide separating
gels and polyacrylamide stacking gels that includes linear
acrylamide as disclosed herein.
[0050] In certain embodiments, the linear acrylamide is added to a
solution of monomeric acrylamide solution, bisacrylamide
crosslinker, initiator(s), and optional catalyzing agent(s),
thereby resulting in the mixture of linear acrylamide, monomeric
acrylamide, bisacrylamide crosslinker, polymerization initiator(s)
and optional catalyst(s) used to make polyacrylamide separating
gels and polyacrylamide stacking gels that includes linear
acrylamide as disclosed herein. In certain embodiments, the linear
acrylamide is added to a solution of monomeric acrylamide,
bisacrylamide crosslinker, ammonium persulfate and
tetramethylethylenediamine (TEMED), thereby resulting in the
mixture used to make polyacrylamide separating gels and
polyacrylamide stacking gels that includes linear acrylamide as
disclosed herein.
[0051] The electrophoresis gels of the compositions, gel cassettes
and methods disclosed herein can be gradient separating gels, in
which the concentration of the polymer (exclusive of the
concentration of any added linear polymer) varies through the
separating gel, generally from low concentration at the top of the
gel body to high concentration at the bottom of the gel body. The
concentration range of the polymer in such gradient separating gels
depends on the application, and in particular the size of the
molecules to be separated. In certain embodiments such separating
gel of the electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein can be gradient
polyacrylamide separating gels having a concentration gradient with
(w/v) acrylamide concentrations ranging from about 2% to about 30%,
from about 2.5% to 25%, from about 3% to about 20%, from about 3%
to about 8%, from about 4% to about 16%, from about 3% to about
12%, from about 4% to about 20%, or from about 5% to about 20%. In
certain embodiments such separating gel of the electrophoresis gels
used in the compositions, gel cassettes and methods disclosed
herein can be gradient polyacrylamide separating gels having a
concentration gradient with (w/v) acrylamide concentrations ranging
from 2% to 30%, from 2.5% to 25%, from 3% to 20%, from 3% to 8%,
from 4% to 16%, from 3% to 12%, from 4% to 20%, or from 5% to
20%.
[0052] The electrophoresis gels of the compositions, gel cassettes
and methods disclosed herein can include both a gradient separating
gel and a stacking gel, wherein the concentration of the stacking
gel polymer is equal to or less than the lowest concentration of
polymer used in the gradient separating gel. In certain
embodiments, the electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein includes both a
polyacrylamide gradient separating gel and a polyacrylamide
stacking gel, wherein the concentration of the acrylamide in the
stacking gel is equal to or less than the lowest concentration of
acrylamide used in the gradient separating gel. In other
embodiments, the electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein includes both a
polyacrylamide gradient separating gel and a polyacrylamide
stacking gel, wherein the concentration of the acrylamide in the
stacking gel is equal to or less than the lowest concentration of
acrylamide used in the gradient separating gel, and the stacking
gel includes linear polyacrylamide at a (w/v) concentration of from
about 0.005% to about 1%, 0.005% to about 0.75%, 0.005% to about
0.5%, 0.005% to about 0.1%, 0.01% to about 1%, 0.01% to about
0.75%, 0.01% to about 0.5%, 0.01% to about 0.1%, 0.02% to about 1%,
0.02% to about 0.75%, 0.02% to about 0.5%, 0.02% to about 0.2% or
0.02% to about 0.1%. In other embodiments, the electrophoresis gels
used in the compositions, gel cassettes and methods disclosed
herein includes both a polyacrylamide gradient separating gel and a
polyacrylamide stacking gel, wherein the concentration of the
acrylamide in the stacking gel is equal to or less than the lowest
concentration of acrylamide used in the gradient separating gel,
and the stacking gel includes linear polyacrylamide at a (w/v)
concentration of from 0.005% to 1%, 0.005% to 0.75%, 0.005% to
0.5%, 0.005% to 0.1%, 0.01% to 1%, 0.01% to 0.75%, 0.01% to 0.5%,
0.01% to 0.1%, 0.02% to 1%, 0.02% to 0.75%, 0.02% to 0.5%, 0.02% to
0.2% or 0.02% to 0.1%. In another embodiment, an electrophoresis
gels used in the compositions, gel cassettes and methods disclosed
herein can include a slab gradient polyacrylamide separating gel
comprising a polyacrylamide concentration of 4%-16%, and a
polyacrylamide stacking gel with a concentration of 3%
polyacrylamide plus 0.05% (weight/volume) of linear
polyacrylamide.
[0053] The gradient separating gels of the electrophoresis gels
used in the compositions, gel cassettes and methods disclosed
herein can also include linear acrylamide present in a (w/v)
concentration of from about 0.005% to about 1%, 0.005% to about
0.75%, 0.005% to about 0.5%, 0.005% to about 0.1%, 0.01% to about
1%, 0.01% to about 0.75%, 0.01% to about 0.5%, 0.01% to about 0.1%,
0.02% to about 1%, 0.02% to about 0.75%, 0.02% to about 0.5%, 0.02%
to about 0.1%, or 0.02% to about 0.1%. In certain embodiments such
gradient separating gels include linear acrylamide present in a
(w/v) concentration of from 0.005% to 1%, 0.005% to 0.75%, 0.005%
to 0.5%, 0.005% to 0.1%, 0.01% to 1%, 0.01% to 0.75%, 0.01% to
0.5%, 0.01% to 0.1%, 0.02% to 1%, 0.02% to 0.75%, 0.02% to 0.5%,
0.02% to 0.1%, or 0.02% to 0.1%. In certain embodiments, such
gradient separating gels are polyacrylamide gradient separating
gels having a concentration gradient with (w/v) acrylamide
concentrations ranging from about 2% to about 30%, from about 2.5%
to 25%, from about 3% to about 20%, from about 3% to about 8%, from
about 4% to about 16%, from about 3% to about 12%, from about 4% to
about 20%, or from about 5% to about 20%. In certain embodiments,
such gradient separating gels are polyacrylamide gradient
separating gels having a concentration gradient with (w/v)
acrylamide concentrations ranging from 2% to 30%, from 2.5% to 25%,
from 3% to 20%, from 3% to 8%, from 4% to 16%, from 3% to 12%, from
4% to 20%, or from 5% to 20%.
[0054] In more specific aspects of the electrophoresis gels used in
the compositions, gel cassettes and methods disclosed herein, the
electrophoresis gels comprise a stacking gel and a separating gel,
in which linear acrylamide is present only in the stacking gel and
is not present in the separating gel. In certain embodiments, such
stacking gels and separating gels both comprise polyacrylamide, but
only the stacking gel comprises linear acrylamide. The addition of
linear acrylamide in the separating gel can potentially affect the
transparency of the separating gel and thereby affect the detection
of sample bands located in the separating gel. Such affects are
also known as "clouding" effects.
[0055] In certain embodiments, the polyacrylamide stacking gel
contains the lowest concentration of acrylamide, with respect to
the acrylamide concentration range of the polyacrylamide separating
gel. In embodiments where the total acrylamide (acrylamide:
bisacrylamide) concentration is below about 3.5% (w/v), the
resulting polyacrylamide matrix is a soft gel that, in addition to
being prone to breakage, may also result in skirting artifacts. The
inclusion of linear polyacrylamide in such low percentage
acrylamide stacking gels can improve the strength of the gels and
may also reduce or eliminate the occurrence of skirting
artifacts.
[0056] The electrophoresis gels used in the compositions, gel
cassettes and methods can include separating gels that are
non-denaturing gels. In certain embodiments such non-denaturing
separating gels comprise linear polyacrylamide, while in other
embodiments such non-denaturing separating gels are polyacrylamide
separating gels that comprise linear polyacrylamide.
[0057] The electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein can include stacking gels
that are non-denaturing gels. In certain embodiments such
non-denaturing stacking gels comprise linear polyacrylamide, while
in other embodiments such non-denaturing stacking gels are
polyacrylamide stacking gels that comprise linear
polyacrylamide.
[0058] The electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein can include both separating
gels and stacking gels that are non-denaturing gels. In certain
embodiments such non-denaturing separating gels and stacking gels
comprise linear polyacrylamide, while in other embodiments such
non-denaturing separating gels and stacking gels are polyacrylamide
separating gels and stacking gels, wherein the stacking gel
comprises linear polyacrylamide. In other embodiments such
non-denaturing separating gels and stacking gels are polyacrylamide
separating gels and stacking gels that comprise linear
polyacrylamide.
[0059] An example of non-denaturing gels used to separate proteins
and protein complexes are Blue native gels ("BN gels"). Such BN
gels have been described by Schagger H and von Jagow G (1991) "Blue
native electrophoresis for isolation of membrane protein complexes
in enzymatically active form" Anal. Biochem. 199: 223-231; Schagger
H, Cramer W A, and von Jagow G (1994) "Analysis of molecular masses
and oligomeric states of protein complexes by blue native
electrophoresis and isolation of membrane protein complexes by
two-dimensional native electrophoresis" Anal. Biochem. 217:
220-230; and Schagger H (2001) "Blue-native gels to isolate protein
complexes from mitochondria" Methods Cell Biol. 65: 231-244, each
of which is herein incorporated by reference in its entireties.
Briefly, in BN gels proteins are stained with Coomassie G-250 which
confers a negative charge to the proteins without denaturing the
proteins. This charge-shifting of proteins by Coomassie G-250
results in proteins being resolved on such "blue-native gels" based
upon their size, thereby making accurate size estimation of native
proteins and protein complexes possible.
[0060] In certain embodiments of the electrophoresis gels used in
the compositions, gel cassettes and methods disclosed herein, such
electrophoresis gels are non-denaturing Blue Native polyacrylamide
gels that include linear polyacrylamide in the stacking gel, and
are used for the separation of proteins and protein complexes. In
other embodiments, the non-denaturing gels, including BN gels,
include gradient separating gels having a concentration gradient
with (w/v) acrylamide concentrations ranging from about 2% to about
30%, from about 2.5% to 25%, from about 3% to about 20%, from about
3% to about 8%, from about 4% to about 16%, from about 3% to about
12%, from about 4% to about 20%, or from about 5% to about 20%. In
other embodiments, the non-denaturing gels, including BN gels,
include gradient separating gels having a concentration gradient
with (w/v) acrylamide concentrations ranging from 2% to 30%, from
2.5% to 25%, from 3% to 20%, from 3% to 8%, from 4% to 16%, from 3%
to 12%, from 4% to 20%, or from 5% to 20%. In addition, such
non-denaturing gels include stacking gels having an acrylamide
(w/v) concentration of from about 1% to about 6% in concentration,
from about 2% to about 5%, or from about 2.5% to about 4%
polyacrylamide. In addition, such non-denaturing gels include
stacking gels having an acrylamide (w/v) concentration of from 1%
to 6% in concentration, from 2% to 5%, or from 2.5% to 4%
polyacrylamide. Such stacking gels also include linear
polyacrylamide at a (w/v) concentration of from about 0.005% to
about 1%, 0.005% to about 0.75%, 0.005% to about 0.5%, 0.005% to
about 0.1%, 0.01% to about 1%, 0.01% to about 0.75%, 0.01% to about
0.5%, 0.01% to about 0.1%, 0.02% to about 1%, 0.02% to about 0.75%,
0.02% to about 0.5%, from about 0.02% to about 0.2%, or 0.02% to
about 0.1%. In certain embodiments, such stacking gels also include
linear polyacrylamide at a (w/v) concentration of from 0.005% to
1%, 0.005% to 0.75%, 0.005% to 0.5%, 0.005% to 0.1%, 0.01% to 1%,
0.01% to 0.75%, 0.01% to 0.5%, 0.01% to 0.1%, 0.02% to 1%, 0.02% to
0.75%, 0.02% to 0.5%, from 0.02% to 0.2%, or 0.02% to 0.1%.
[0061] In certain embodiments the separating gel, which can be a
gradient gel, does not include linear acrylamide.
[0062] The electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein can be denaturing gels,
wherein the gels includes a detergent(s), chaotropic agent(s) or
combinations thereof. Chaotropic agents include, but are not
limited to, sodium trifluoroacetate, sodium perchlorate, sodium
iodide, urea, guanidinium chloride and guanidine isothiocyanate.
Denaturing detergents include, but are not limited to, sodium
dodecyl sulfate (SDS).
[0063] The electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein can include separating gels
that are denaturing gels. In certain embodiments such denaturing
separating gels comprise linear polyacrylamide, while in other
embodiments such denaturing separating gels are polyacrylamide
separating gels that comprise linear polyacrylamide.
[0064] The electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein can include stacking gels
that are denaturing gels. In certain embodiments such denaturing
stacking gels comprise linear polyacrylamide, while in other
embodiments such denaturing stacking gels are polyacrylamide
stacking gels that comprise linear polyacrylamide.
[0065] The electrophoresis gels used in the compositions, gel
cassettes and methods disclosed herein can include both separating
gels and stacking gels that are denaturing gels. In certain
embodiments such denaturing separating gels and stacking gels
comprise linear polyacrylamide, while in other embodiments such
denaturing separating gels and stacking gels are polyacrylamide
separating gels and stacking gels, wherein the stacking gel
comprises linear polyacrylamide. In other embodiments such
denaturing separating gels and stacking gels are polyacrylamide
separating gels and stacking gels that comprise linear
polyacrylamide.
B. Gel Cassettes Having Consistent Plate-to Plate Spacing Along
their Length
[0066] Another aspect of the methods and compositions disclosed
herein are gel cassettes that have a consistent internal gap
between the front and back plates. For purposes of illustration,
FIG. 2 shows a cassette (21) that has been cut down the middle, in
which the gap (24) between the back plate (23) and the front plate
(22) is substantially the same between a point along the upper edge
(25) of the cassette that is in the middle of the cassette (27) and
a point along the upper edge (25) of the cassette that is at the
outer edge of the cassette (26). A "consistent internal gap between
the front and back plates" of a cassette means that the space
within a cassette that holds the gel has substantially the same
front-to-back depth throughout the space containing the gel. That
is, the front-to-back depth at the top edge of the space (the end
of the cassette where a comb is inserted to form wells) is
substantially the same as the front-to-back depth in the mid-region
of the space, the bottom region of the space and the outer edges of
the space.
[0067] As used herein, "substantially the same" or "substantially
equal" means that the internal gap width between plates at the top
edge of the cassette (at the end of the cassette where a comb can
be inserted) does not vary by more than about 5% of the greatest
gap width between an edge of the cassette and the central region of
the cassette, and preferably does not vary by more than about 2% of
the internal width between the two plates from the edges of the
cassette to the mid region of the cassette. In some preferred
embodiments, the gap width between the front and back plate does
not vary by more than about 1% across the top of the cassette (or
the region of the cassette corresponding to where a comb can be
inserted to form sample wells). For example, a cassette may be
designed to hold a gel of 1 mm thickness, which corresponds to the
internal gap width of the cassette. In this case, the width of the
internal space of the cassette does not vary by more than about
0.05 mm, by more than about 0.02 mm, or by more than about 0.01 mm
across the top of the cassette (or the region of the cassette
corresponding to where a comb is inserted). In another example, a
cassette may be designed to hold a gel of 1.5 mm thickness. In this
case, the width of the internal space of the cassette does not vary
by more than about 0.075 mm, by more than about 0.03 mm, or by more
than about 0.015 mm across the top of the cassette (or the region
of the cassette corresponding to where a comb is inserted).
[0068] In certain embodiments, the gel cassette has an internal gap
between the front and back plates in which the internal distance
between the front and back plates does not substantially vary along
the upper edge of the cassette.
[0069] A gel cassette can have front and back plates constructed of
any suitable material, where suitable materials include plastics,
polymers, glass, ceramics, or any material that is not permeable to
fluids and is non-conducting under standard electrophoresis
conditions. The gel cassettes disclosed herein can be made of a
polymer which is transparent to visible light, transparent to
ultraviolet light, transparent to infra-red light, or transparent
to both visible and ultraviolet light. Non-limiting examples of
polymers used to make the gel cassettes disclosed herein are
styrene acrylonitrile (SAN), polycarbonate, polystyrene, acrylic
based polymers, polymethyl methacrylate (PMMA), polyethylene
terephthalate (PET), glycol-modified polyethylene terephthalate
(PETG), polypropylene, Acetel and copolymers thereof. The plates of
the gel cassettes disclosed herein can be coated on the gel-facing
side with one or more polymers such as, by way of example only,
latex, thereby preventing sticking of the gel to the plates when
the gel is to be removed after electrophoresis. The gel cassettes
or the plates that will be attached to form a gel cassette,
disclosed herein, may be fabricated using molding techniques, hot
embossing methods, casting processes, thermoforming methods,
stereolithography processes, machining methods and milling
processes. In further or alternative embodiments, such molding
techniques include injection molding and compression molding.
[0070] The front and back plates can be attached to one another (to
form a gel cassette) by any feasible means including, but not
limited to, being molded as a single piece along with edge pieces
that connect the front and back plates at the side, being welded
together (by way of example only, ultrasonic welding), being
fastened together with adhesives, being thermally treated, or being
held with attachment means screws, pins, snaps, or clamps. In
certain embodiments of the gel cassettes disclosed herein, either
or both of the front and back plates of the cassette have raised
borders around the edges where the cassettes are attached, by way
of example only, by welding, that provide spacers that establish
the distance between the attached plates. In some embodiments, the
plates so designed are welded together to specifications such that
the spacer thickness establishing the distance between plates of
the cassette is substantially the same from the outer edges of the
cassette to at least the midpoint of the cassette along the upper
edge of the cassette.
[0071] The gel cassette disclosed herein can be of any size used in
any electrophoresis system. The dimensions of a gel cassette having
a consistent gap width are not limiting and include, but are not
limited to, gel cassette having a front plate and back plates from
about 5 cm to about 30 cm in width, from about 5 cm to about 60 cm
in length, and from about 1 mm to about 5 mm in plate thickness. In
some embodiments, the plates of the gel cassette can be about 4 mm
thick or less, about 3 mm thick or less, about 2.5 mm thick or
less, about 2 mm thick or less, about 1.5 mm thick or less, or
about 1 mm thick or less. In other embodiments, the back plate of a
cassette is between 2.5 mm and 3 mm thick in the area containing
the gel, and the front plate of a cassette is between 1.5 and 2 mm
thick in the area containing the gel.
[0072] In the gel cassettes disclosed herein the front plate and
back plate need not be of equal dimensions. In addition, the front
plate, the back plate, or both, can be irregularly shaped along one
or more sides such as, by way of example only, having at least a
portion of an outer edge that is inset or curved. The gap width
between plates of an assembled cassette can be from about 0.1 mm to
about 10 mm, from about 0.1 mm to about 5 mm, from about 0.25 mm to
about 5 mm, from about 0.25 mm to about 3 mm, from about 0.25 mm to
about 2.5 mm, from about 0.25 mm to about 2 mm, from about 0.25 mm
to about 1.5 mm, from about 0.25 mm to about 1 mm, from about 0.5
mm to about 5 mm, from about 0.5 mm to about 3 mm, from about 0.5
mm to about 2.5 mm, from about 0.5 mm to about 2 mm, from about 0.5
mm to about 1.5 mm, or from about 0.5 mm to about 1 mm. In
illustrative embodiments, the gap width between plates of an
assembled cassette is from 0.1 mm to 10 mm, from 0.1 mm to 5 mm,
from 0.25 mm to 5 mm, from 0.25 mm to 3 mm, from 0.25 mm to 2.5 mm,
from 0.25 mm to 2 mm, from 0.25 mm to 1.5 mm, from 0.25 mm to 1 mm,
from 0.5 mm to 5 mm, from 0.5 mm to 3 mm, from 0.5 mm to 2.5 mm,
from 0.5 mm to 2 mm, from 0.5 mm to 1.5 mm, or from 0.5 mm to 1 mm.
The gap width can be established by spacers between the plates
along the outer edges of the plates (top and bottom), or by border
regions of the plates (top and bottom) that can be fastened
together using welding methods (by way of example only, ultrasonic
welding), thermal treatment, adhesives, gaskets, clamps, or
fasteners. In certain embodiments, the cassette plates are made of
one or more plastics such as, for example, and one or both of the
back plate or the front plate has a raised border region that is
welded or heat fused to the partner plate, and the welding process
in part determines the gap width by determining the thickness of
the border region that remains between the welded or heat fused
plates of the cassette.
[0073] A non-limiting example of a gel cassette having a consistent
internal gap between the front and back plates has front and back
plates that are 10 cm.times.10 cm, where the front plate is about
1.75 mm thick and the back plate is about 2.55 mm thick, and the
two plates are welded together such that the gap width of the
cassette in the region where a comb is to be inserted to form wells
is consistently about 1 mm.
[0074] Another non-limiting example of a gel cassette having a
consistent internal gap between the front and back plates has front
and back plates that are 10 cm.times.10 cm, where the front and
back plates are about 1.75 mm and about 2.5 mm thick, respectively,
and the plates welded together such that the gap width of the
cassette is consistently about 1.5 mm.
[0075] Another non-limiting example of a gel cassette having a
consistent internal gap between the front and back plates has front
and back plates that are 15 cm.times.15 cm, where the front and
back plates are about 1.75 mm and about 2.55 mm thick,
respectively, and the two plates are welded together such that the
gap width of the cassette in the region where a comb is to be
inserted to form wells is consistently about 1 mm.
[0076] Another non-limiting example of a gel cassette having a
consistent internal gap between the front and back plates has front
and back plates that are 15 cm.times.15 cm, where the front and
back plates are about 1.75 mm and about 2.55 mm thick,
respectively, and the two plates are welded together such that the
gap width of the cassette in the region where a comb is to be
inserted to form wells is consistently about 1.5 mm.
[0077] These examples of gel cassettes having a consistent internal
gap between the front and back plates are for illustrative purposes
only and are not intended to be limiting in any way.
[0078] The gel cassettes disclosed herein also include gel
cassettes having a consistent gap width across the upper edge of
the cassette where sample loading occurs, wherein the gap width
varies by less than about 5%, in some preferred embodiments by no
more than about 2% or by no more than about 1%.
[0079] The gel cassettes with a consistent gap widths disclosed
herein can contain a gel that comprises any suitable gel forming
polymer, including, but not limited to, synthetic polymers, natural
polymers and combinations thereof. Examples of such synthetic
polymers include, but are not limited to, linear polyacrylamide,
crosslinked polyacrylamide, or combinations thereof. Examples of
such natural polymers include, but are not limited to,
polysaccharides such as agarose. In certain embodiments such gels
can comprise agarose, polyacrylamide, or combinations of agarose
and polyacrylamide.
[0080] The gel cassettes with a consistent gap widths disclosed
herein can contain any stacking gel and/or separating gel disclosed
herein. In certain embodiments, the gel cassettes contain gels that
includes linear acrylamide, as described herein. When present in a
gel that includes a stacker, linear acrylamide can be present in
the stacker and not in the separating gel. In alternate
embodiments, linear acrylamide can be present in the stacker and in
the separating gel. The gel can be of any polymer concentration as
disclosed herein including, by way of example only, from about 0.3%
to about 3% in the case of agarose, or from about 1% to about 30%
acrylamide. When present, the linear acrylamide can be present at
any (w/v) concentration disclosed herein including, but not limited
to, from about 0.005% to about 1% and from about 0.01% to about
0.5%. The separating gel in gel cassettes with a consistent gap
widths disclosed herein can be a gradient gel as disclosed herein.
The separating gel in gel cassettes with a consistent gap widths
disclosed herein can optionally include a stacking gel as disclosed
herein, where the stacking gel has a lower concentration of gel
polymer than that in the separating gel. Where combination gels are
used, optimal concentrations of each component can be determined
empirically or as guided by published protocols.
[0081] A gel contained in a gel cassette as described herein can
also include at least one of the following: one or more buffers,
salts, reducing agents, oxidizing agents, alkylating agents,
denaturants, chelators, polymers, or detergents. In some
embodiments, a gel contained in such cassettes is a gel to be used
for separation of nucleic acids. In some embodiments, a gel
contained in such cassettes is a gel to be used for polypeptide
electrophoresis. In some embodiments, a gel contained in such
cassettes is used for native electrophoresis of proteins, in which
proteins and protein complexes are not denatured prior to or during
electrophoresis. In other embodiments, a gel contained in such
cassette is a gel to be used for polypeptide electrophoresis. In
some embodiments, a gel contained in such cassettes is used for
native electrophoresis of proteins, in which proteins and protein
complexes are not denatured prior to or during electrophoresis and
the gel and the running buffer(s) do not include denaturants, such
as but not limited to denaturing detergents, urea, formamide,
chaotropes, and the like. In a non-limiting example, Blue Native
gels are used in cassettes described herein having a consistent
internal gap width. In certain embodiments, the Blue Native gels
have stacking gels. In other embodiments, the Blue Native gels have
stacking gels that include linear acrylamide.
[0082] The buffer or buffers included in gels that are contained in
gel cassettes as described herein can be any electrophoresis
buffer, including but not limited zwitterionic buffers. In certain
embodiments the gel buffer has a pH between 5 and 9 at ambient
temperature. In certain embodiments the gel buffer has a pH between
6 and 8.5 at ambient temperature. In certain embodiments the gel
buffer has a pH between 6 and 8 at ambient temperature. In certain
embodiments the gel buffer has a pH between 6 and 7 at ambient
temperature. In certain embodiments the gel buffer has a pH between
7 and 8 at ambient temperature. In certain embodiments the gel
buffer has a pH between 5 and 9 at 25.degree. C. In certain
embodiments the gel buffer has a pH between 6 and 8.5 at 25.degree.
C. In certain embodiments the gel buffer has a pH between 6 and 8
at 25.degree. C. In certain embodiments the gel buffer has a pH
between 7 and 8 at 25.degree. C. In certain embodiments the gel
buffer has a pH between 6 and 7 at 25.degree. C.
[0083] In certain embodiments the buffer or buffers included in
gels that are contained in gel cassettes as described herein
comprises a buffer having a pKa between about 5 and about 8.5 at
ambient temperature. In certain embodiments the gel buffer
comprises a buffer having a pKa between about 6 and about 8.5 at
ambient temperature. In certain embodiments the gel buffer
comprises a buffer having a pKa between about 6 and about 8 at
ambient temperature. In certain embodiments the gel buffer
comprises a buffer having a pKa between about 6 and about 7 at
ambient temperature. In certain embodiments the gel buffer
comprises a buffer having a pKa between about 7 and about 8 at
ambient temperature. In certain embodiments the gel buffer
comprises a buffer having a pKa between about 5 and about 8.5 at
25.degree. C. In certain embodiments the gel buffer comprises a
buffer having a pKa between about 6 and about 8.5 at 25.degree. C.
In certain embodiments the gel buffer comprises a buffer having a
pKa between about 6 and about 8 at 25.degree. C. In certain
embodiments the gel buffer comprises a buffer having a pKa between
about 6 and about 7 at 25.degree. C. In certain embodiments the gel
buffer comprises a buffer having a pKa between about 7 and about 8
at 25.degree. C.
[0084] The buffer or buffers included in gels that are contained in
gel cassettes as described herein include, but are not limited to,
succinate, citrate, borate, maleate, cacodylate,
N-(2-Acetamido)iminodiacetic acid (ADA),
2-(N-morpholino)-ethanesulfonic acid (MES),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),
piperazine-N,N'-2-ethanesulfonic acid (PIPES),
2-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES),
3-(N-morpholino)-propanesulfonic acid (MOPS),
N-tris-(hydroxymethyl)-2-ethanesulfonic acid (TES),
N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES),
3-(N-tris-(hydroxymethyl) methylamino)-2-hydroxypropanesulfonic
acid (TAPSO),
3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid
(DIPSO), N-(2-Hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic
acid) (HEPPSO), 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid
(EPPS), N-[Tris(hydroxymethyl)methyl]glycine (Tricine),
N,N-Bis(2-hydroxyethyl) glycine (Bicine),
(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic
acid (TAPS),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), tris (hydroxy methyl) amino-methane (Tris),
TRIS-Acetate-EDTA (TAE), glycine,
bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane (BisTris), or
combinations thereof. In addition, such gel buffers can include
ethylene diamine tetraacetic acid (EDTA).
[0085] The concentration of the buffer or buffers included in gels
that are contained in gel cassettes as described herein can be from
about 10 mM to about 1.5 M. In certain embodiments the
concentration can be between about 10 mM and about 1 M. In certain
embodiments the concentration can be between about 20 mM and about
500 mM, and in other embodiments the concentration is between about
50 mM and about 300 mM. In certain embodiments the concentration
can be between about 10 mM and about 200 mM, and in other
embodiments the concentration is between about 10 mM and about 500
mM. In certain embodiments the concentration can be between about
50 mM and about 200 mM, and in other embodiments the concentration
is between about 50 mM and about 500 mM. In certain embodiments the
concentration can be between about 5 mM and about 200 mM, and in
other embodiments the concentration is between about 5 mM and about
500 mM. In certain embodiments the concentration can be between
about 5 mM and about 1 M. In certain embodiments, the concentration
of the buffer or buffers included in gels that are contained in gel
cassettes as described herein can be from 10 mM to 1.5 M. In
certain embodiments the concentration can be between 10 mM and 1 M.
In certain embodiments the concentration can be between 20 mM and
500 mM, and in other embodiments the concentration is between 50 mM
and 300 mM. In certain embodiments the concentration can be between
10 mM and 200 mM, and in other embodiments the concentration is
between 10 mM and 500 mM. In certain embodiments the concentration
can be between 50 mM and 200 mM, and in other embodiments the
concentration is between 50 mM and 500 mM. In certain embodiments
the concentration can be between 5 mM and 200 mM, and in other
embodiments the concentration is between 5 mM and 500 mM. In
certain embodiments the concentration can be between 5 mM and 1
M.
[0086] Various gel cassettes have been described in U.S. Pat. No.
7,122,104, U.S. Pat. No. 6,562,213, U.S. Pat. No. 5,582,702, U.S.
Pat. No. 5,865,974, U.S. Pat. No. 6,379,516, U.S. patent
application Ser. No. 11/470,308 and patent application Ser. No.
10/056,050, each of which is herein incorporated by reference in
their entirety. Such cassettes including, but are not limited to,
multiwell cassettes, can contain the electrophoresis gels disclosed
herein and be used in the compositions and methods described
herein.
[0087] The electrophoresis gels described herein can be used to
separate components of a sample including, but not limited to,
separating biomolecules. The methods of separating samples
components on such electrophoresis gels includes, but are not
limited to, applying one or more samples to an electrophoresis gel
and electrophoretically separating the sample components on the
electrophoresis gel. In certain embodiments, such methods include,
but are not limited to, applying one or more samples comprising one
or more biomolecules to an electrophoresis gel that comprises a
separating gel, and electrophoretically separating one or more
biomolecules or biomolecular complexes on the separating gel. In
certain embodiments, such methods include, but are not limited to,
applying one or more samples comprising one or more biomolecules to
an electrophoresis gel that comprises a separating gel comprising
linear polyacrylamide, and electrophoretically separating one or
more biomolecules or biomolecular complexes on the separating gel.
In certain embodiments, such methods include, but are not limited
to, applying one or more samples comprising one or more
biomolecules to an electrophoresis gel that comprises a stacking
gel portion and a separating gel portion, and electrophoretically
separating one or more biomolecules or biomolecular complexes on
the separating gel. In other embodiments, such methods include, but
are not limited to, applying one or more samples comprising one or
more biomolecules to an electrophoresis gel that comprises a
stacking gel portion that comprises linear polyacrylamide and a
separating gel portion, and electrophoretically separating one or
more biomolecules or biomolecular complexes on the separating gel.
In certain other embodiments, such methods include, but are not
limited to, applying one or more samples comprising one or more
biomolecules to an electrophoresis gel that comprises a stacking
gel portion and a separating gel portion, wherein both the stacking
gel and the separating gel comprise linear polyacrylamide; and
electrophoretically separating one or more biomolecules or
biomolecular complexes on the separating gel.
[0088] In practicing the methods of the invention, wherein the
stacking gel comprises linear polyacrylamide, the presence of
skirting bands on the separating gel is reduced. In practicing the
methods of the invention, wherein the separating gel comprises
linear polyacrylamide, the presence of skirting bands on the
separating gel is reduced. In practicing the methods of the
invention, wherein the stacking gel and the separating gel comprise
linear polyacrylamide, the presence of skirting bands on the
separating gel is reduced. By "reduced" is meant the appearance,
intensity, or width of skirting bands is less on electrophoresis
gels having linear polyacrylamide than in electrophoresis gels
without linear polyacrylamide run under the same conditions and
having the same composition (except having linear
polyacrylamide).
[0089] The gels used in such methods can be any electrophoresis gel
described herein. In certain embodiments the gels are
polyacrylamide gels. In other embodiments the gels are agarose
gels, while in other embodiments the gels comprise both acrylamide
and agarose. The gels used in such methods can be denaturing gels
as disclosed herein, including, but not limited to, SDS
polyacrylamide gels. The gels used in such methods can be
non-denaturing gels as disclosed herein, including, but not limited
to, blue native (BN) gels. The separating gel used in such methods
can have any suitable composition. In some embodiments, the
separating gel used in such methods includes linear polyacrylamide.
In other embodiments, the separating gel used in such methods does
not include linear polyacrylamide. In certain embodiments, the
separating gel used in such methods comprises polyacrylamide, while
in other embodiments the separating gel used in such methods
comprises both polyacrylamide and linear polyacrylamide. In some
embodiments, the separating gel used in such methods is a gradient
gel. In certain embodiments, the electrophoresis gels used in such
methods are multiwell gels, in which two or more samples, one or
more samples, or multiple loadings of the same sample are
electrophoresed simultaneously. In certain embodiments, the
electrophoresis gels used in such methods are multiwell gels, in
which one or more molecular weight standards along with two or more
samples, one or more samples, or multiple loadings of the same
sample are electrophoresed simultaneously.
[0090] Native or non-denaturing gels used in the methods disclosed
herein are run without denaturing agents such as, for example,
protein-denaturing detergents or chaotropes in the gel or in the
running buffer(s). The native gels used in the methods disclosed
herein include, but are not limited to, blue native (BN) gels. The
use of blue native gels, in which the cathode buffer, the protein
sample buffer, or both, contain Coomassie G-250 has been described
in Schagger H and von Jagow G (1991) "Blue native electrophoresis
for isolation of membrane protein complexes in enzymatically active
form" Anal Biochem. 199: 223-231; Schagger H, Cramer W A, and von
Jagow G (1994) "Analysis of molecular masses and oligomeric states
of protein complexes by blue native electrophoresis and isolation
of membrane protein complexes by two-dimensional native
electrophoresis" Anal Biochem. 217: 220-230; and Schagger H (2001)
"Blue-native gels to isolate protein complexes from mitochondria"
Methods Cell Biol. 65: 231-244. The Coomassie G-250 binds proteins
in their native state, thereby conferring a negative charge to the
proteins. The negatively charged Coomassie stained proteins then
migrate to the anode at a velocity that is proportional to their
charge and molecular weight.
[0091] The biomolecules separated on an electrophoresis gel
described herein and using the methods described herein can be any
biomolecule, including, but not limited to, proteins, nucleic
acids, polysaccharides, lipids, and other macromolecules. In
certain embodiments the biomolecules separated on an
electrophoresis gel described herein and using the methods
described herein are proteins, nucleic acids, or biomolecular
complexes that include proteins or nucleic acids. By way of example
only, such biomolecular complexes can be any combination of
associated proteins, peptides, nucleic acids, and polysaccharides.
In exemplary embodiments, the biomolecules or complexes separated
on an electrophoresis gel that comprises linear polyacrylamide in
the stacking portion of the gel are proteins or molecular complexes
that include proteins. In certain embodiments the electrophoresis
gel has two or more wells for electrophoresis of at least two
standards or at least one sample and at least one molecular weight
standard. In exemplary embodiments, the biomolecules or complexes
separated on an electrophoresis gel, that is contained in a gel
cassette having a consistent internal gap as disclosed herein, are
proteins or molecular complexes that include proteins.
[0092] The one or more samples, or two or more replicates of the
same sample, applied to the electrophoresis gel can be any samples
that include biomolecules, and can be environmental samples, tissue
samples, cell extracts or fractions, etc. The samples can be crude
samples such as lysates, fractionated samples, or partially or
substantially processed or purified samples. Prior to loading on an
electrophoresis gel, a sample can be treated with solubilizers,
reducing agents, denaturing agents or treatments, detergents,
chaotropic agent, or other sample preparation agents. In the case
of Blue Native gel electrophoresis, proteins and protein complexes
are not denatured prior to electrophoresis, but may be exposed to
solubilizers such as, for example, non-denaturing detergents.
[0093] The methods, gels and gel cassettes described herein can be
used to electrophoretically separate biomolecules and/or
biomolecular complexes. However, general electrophoresis methods
and parameters, such as sample loading and electrophoresis run time
are known in the art and are well within the capabilities of a
killed artisan. In addition, apparatuses designed to hold gel
assemblies, gel cassettes and running buffers, during
electrophoresis are well known in the art and widely available
commercially, including but not limited to, the SureLock.TM.
mini-cell electrophoresis apparatus (Invitrogen Corp, Carlsbad,
Calif.). Such apparatuses can be used with the methods, gels and
gel cassettes described herein.
[0094] Although electrophoresis conditions can be determined by a
practitioner guided by protocols known in the art. The
electrophoretic separations disclosed herein can be achieved using
constant voltage, pulsed voltage, step-gradient voltage, constant
current, pulsed current, step-gradient current, constant power,
step-gradient power or pulsed power. Subsequently, the applied
electric field (V/cm) in the methods disclosed herein can be
constant or pulsed. It is understood that the magnitude of the
applied voltage, applied current or applied power to achieve the
electric fields ranges provided below will vary depending on the
dimensions of the electrophoresis cassette and buffer conductivity.
By way of example only, the applied voltage can range from 5V to
2000V, and in certain embodiments the applied voltage can range
from 5V to 1000V, 5V to 500V, 5V to 250V, or 5V to 100V. In other
embodiments, the applied voltage can range from about 10 to about
1,000 V, from about 25 to about 750 V, or from about 40 to about
300 V. By way of example only, the applied current can range from 5
mA to 400 mA, and in certain embodiments the applied current can
range from 5 mA to 200 mA, 5 mA to 100 mA, 5 mA to 50 mA, or 5 mA
to 25 mA. In one embodiment the applied current can be 15 mA. By
way of example only, the applied current can range from 5 mA to 400
mA, and in certain embodiments the applied power can range from 25
mW to 400 W, 25 mW to 100 W, 25 mW to 50 W, or 25 mW to 25 W. In
one embodiment the applied power can be 4.5 W. In addition the
polarity of the applied voltage (constant or pulsed) can be
positive or negative, and the polarity of the applied current
(constant or pulsed) can be positive or negative.
[0095] In certain embodiments the magnitude of the constant
electric field applied is between 1 V/cm and 100 V/cm. In certain
embodiments the magnitude of the constant electric field applied is
between 1 V/cm and 50 V/cm. In certain embodiments the magnitude of
the constant electric field applied is between 1 V/cm and 25 V/cm.
In certain embodiments the magnitude of the constant electric field
applied is between 1 V/cm and 15 V/cm. In certain embodiments the
magnitude of the constant electric field applied is between 1 V/cm
and 10 V/cm.
[0096] For the application of a step-gradient voltage, the step
profile can be a single step, two steps, or greater than two steps.
The step voltage is applied to a constant baseline electric field,
established by applying a baseline voltage, and the magnitude of
this baseline electric field is from 0 V/cm to 100 V/cm. In certain
embodiments the magnitude of this baseline electric field is from 0
V/cm to 50 V/cm. In certain embodiments the magnitude of this
baseline electric field is from 0 V/cm to 25 V/cm. In certain
embodiments the magnitude of this baseline electric field is from 0
V/cm to 10 V/cm. In certain embodiments the magnitude of the
baseline voltage is from 0V to 1000V, while in other embodiments
the magnitude of the baseline voltage is from 0V to 500V. In
certain embodiments the magnitude of the baseline voltage is from
0V to 200V, while in other embodiments the magnitude of the
baseline voltage is from 0V to 100V. In certain embodiments the
magnitude of the baseline voltage is from 0V to 50V, while in other
embodiments the magnitude of the baseline voltage is from 0V to
10V. The magnitude of the voltage step applied to the baseline
voltage can be from 10V to 2000V, while in other embodiments the
voltage step is from 10V to 1000V. In certain embodiments the
magnitude of the voltage step applied to the baseline voltage is
from 10V to 500V, while in other embodiments the magnitude of the
voltage step is from 10V to 200V. In certain embodiments the
magnitude of the voltage step applied to the baseline voltage is
from 10V to 100V, while in other embodiments the magnitude of the
voltage step is from 10V to 50V. For multiple steps, such as two or
more steps, the magnitude of each step can be symmetric (i.e. the
same), or the magnitude of each step can be asymmetric (i.e.
different). In certain embodiments a two step symmetric
step-gradient voltage profile is a first 50V step applied to a 0V
baseline, followed by another 50V step. In certain embodiments a
two step asymmetric step-gradient voltage profile is a first 50V
step applied to a 0V baseline, followed by a 450V step. In certain
embodiments a two step asymmetric step-gradient voltage profile is
a first 50V step applied to a 0V baseline, followed by a 500V step.
In other embodiments a two step asymmetric step-gradient voltage
profile is a first 75V step applied to a 0V baseline, followed by a
175V step. In other embodiments a two step asymmetric step-gradient
voltage profile is a first 75V step applied to a 0V baseline,
followed by a 250V step. A single step, or independently each step
of a multiple step-gradient, can be run for from about 5 minutes to
about 500 minutes, depending on the magnitude of the applied
voltages. In certain embodiments the step run times can be from
about 5 minutes to about 150 minutes. In certain embodiments the
step run times can be from about 5 minutes to about 100 minutes. In
certain embodiments the step run times can be from about 5 minutes
to about 60 minutes. In certain embodiments the step run times can
be from about 5 minutes to about 30 minutes. In certain embodiments
for a two step asymmetric step-gradient voltage profile the first
step is applied for 15 minutes and the second step is applied for
45 minutes. In certain embodiments for a two step asymmetric
step-gradient voltage profile the first step is applied for 15
minutes and the second step is applied for 50 minutes. In certain
embodiments for a two step asymmetric step-gradient voltage profile
the first step is applied for 15 minutes and the second step is
applied for 55 minutes. In certain embodiments for a two step
asymmetric step-gradient voltage profile the first step is applied
for 15 minutes and the second step is applied for 60 minutes. In
certain embodiments for a two step asymmetric step-gradient voltage
profile the first step is applied for 15 minutes and the second
step is applied for 65 minutes.
[0097] For the application of a step-gradient current profile, the
step profile can be a single step, two steps, or greater than two
steps. The step current is applied to a constant baseline electric
field, established by applying a baseline current, and the
magnitude of this baseline electric field is from 0 V/cm to 100
V/cm. In certain embodiments the magnitude of this baseline
electric field is from 0 V/cm to 50 V/cm. In certain embodiments
the magnitude of this baseline electric field is from 0 V/cm to 25
V/cm. In certain embodiments the magnitude of this baseline
electric field is from 0 V/cm to 10 V/cm. In certain embodiments
the magnitude of the baseline current is from 0 mA to 10 mA, while
in other embodiments the magnitude of the baseline current is from
0 mA to 5 mA. In certain embodiments the magnitude of the baseline
current is from 0 mA to 2 mA, while in other embodiments the
magnitude of the baseline current is from 0 mA to 1 mA. In certain
embodiments the magnitude of the baseline current is from 0 mA to
0.5 mA. The magnitude of the current step applied to a baseline
current can be from 0.5 mA to 100 mA, while in other embodiments
the current step is from 0.5 mA to 50 mA. In certain embodiments
the magnitude of the current step applied to a baseline current is
from 0.5 mA to 25 mA, while in other embodiments the magnitude of
the current step is from 0.5 mA to 10 mA. In certain embodiments
the magnitude of the current step applied to the baseline current
is from 0.5 mA to 5 mA, while in other embodiments the magnitude of
the current step is from 0.5 mA to 2 mA. For multiple steps, such
as two or more steps, the magnitude of each step can be symmetric
(i.e. the same), or the magnitude of each step can be asymmetric
(i.e. different). In certain embodiments a two step symmetric
step-current current profile is a first 7 mA step applied to a 0 mA
baseline, followed by another 7 mA step. In certain embodiments a
two step asymmetric step-gradient current profile is a first 1 mA
step applied to a 0 mA baseline, followed by a 14 mA step. A single
step, or independently each step of a multiple step-gradient, can
be run for from about 5 minutes to about 500 minutes, depending on
the magnitude of the applied current. In certain embodiments the
step run times can be from about 5 minutes to about 150 minutes. In
certain embodiments the step run times can be from about 5 minutes
to about 100 minutes. In certain embodiments the step run times can
be from about 5 minutes to about 60 minutes. In certain embodiments
the step run times can be from about 5 minutes to about 30 minutes.
In certain embodiments for a two step asymmetric step-gradient
current profile the first step is applied for 15 minutes and the
second step is applied for 45 minutes. In certain embodiments for a
two step asymmetric step-gradient current profile the first step is
applied for 15 minutes and the second step is applied for 50
minutes. In certain embodiments for a two step asymmetric
step-gradient current profile the first step is applied for 15
minutes and the second step is applied for 55 minutes. In certain
embodiments for a two step asymmetric step-gradient current profile
the first step is applied for 15 minutes and the second step is
applied for 60 minutes. In certain embodiments for a two step
asymmetric step-gradient current profile the first step is applied
for 15 minutes and the second step is applied for 65 minutes.
[0098] For the application of a step-gradient power profile, the
step profile can be a single step, two steps, or greater than two
steps. The step current is applied to a constant baseline electric
field, established by applying a baseline power level, and the
magnitude of this baseline electric field is from 0 V/cm to 100
V/cm. In certain embodiments the magnitude of this baseline
electric field is from 0 V/cm to 50 V/cm. In certain embodiments
the magnitude of this baseline electric field is from 0 V/cm to 25
V/cm. In certain embodiments the magnitude of this baseline
electric field is from 0 V/cm to 10 V/cm. In certain embodiments
the magnitude of the baseline power is from 0 W to 10 W, while in
other embodiments the magnitude of the baseline power is from 0 W
to 5 W. In certain embodiments the magnitude of the baseline power
is from 0 W to 2 W, while in other embodiments the magnitude of the
baseline current is from 0 W to 1 W. In certain embodiments the
magnitude of the baseline power is from 0 W to 0.5 W. The magnitude
of the power step applied to a baseline can be from 0.5 W to 10 W,
while in other embodiments the power step is from 0.5 W to 5 W. In
certain embodiments the magnitude of the power step applied to a
baseline is from 0.5 W to 2 W, while in other embodiments the
magnitude of the power step is from 0.5 mA to 1 W. For multiple
steps, such as two or more steps, the magnitude of each step can be
symmetric (i.e. the same), or the magnitude of each step can be
asymmetric (i.e. different). In certain embodiments a two step
symmetric step-power profile is a first 1.5 W step applied to a 0 W
baseline, followed by another 1.5 W step. In certain embodiments a
two step asymmetric step-gradient power profile is a first 0.5 W
step applied to a 0 mA baseline t, followed by a 3 W step. A single
step, or independently each step of a multiple step-gradient, can
be run for from about 5 minutes to about 500 minutes, depending on
the magnitude of the applied current. In certain embodiments the
step run times can be from about 5 minutes to about 150 minutes. In
certain embodiments the step run times can be from about 5 minutes
to about 100 minutes. In certain embodiments the step run times can
be from about 5 minutes to about 60 minutes. In certain embodiments
the step run times can be from about 5 minutes to about 30 minutes.
In certain embodiments for a two step asymmetric step-gradient
power profile the first step is applied for 15 minutes and the
second step is applied for 45 minutes. In certain embodiments for a
two step asymmetric step-gradient power profile the first step is
applied for 15 minutes and the second step is applied for 50
minutes. In certain embodiments for a two step asymmetric
step-gradient power profile the first step is applied for 15
minutes and the second step is applied for 55 minutes. In certain
embodiments for a two step asymmetric step-gradient power profile
the first step is applied for 15 minutes and the second step is
applied for 60 minutes. In certain embodiments for a two step
asymmetric step-gradient power profile the first step is applied
for 15 minutes and the second step is applied for 65 minutes.
[0099] The profile of the pulsed electric field (applied voltage,
current or power) can be a square wave, triangular wave or sine
wave, and such profiles can be symmetric or asymmetric. The pulsed
electric field is applied to a constant baseline electric field and
the magnitude of this baseline electric field is from 0 V/cm to 100
V/cm. In certain embodiments the magnitude of this baseline
electric field is from 0 V/cm to 50 V/cm. the magnitude of this
baseline electric field is from 0 V/cm to 25 V/cm. the magnitude of
this baseline electric field is from 0 V/cm to 10 V/cm. In certain
embodiments the magnitude of the pulsed electric field applied in
addition to the baseline electric field is between 1 V/cm and 100
V/cm. In certain embodiments the magnitude of the pulsed electric
field applied in addition to the baseline electric field is between
1 V/cm and 50 V/cm. In certain embodiments the magnitude of the
pulsed electric field applied in addition to the baseline electric
field is between 1 V/cm and 25 V/cm. In certain embodiments the
magnitude of the pulsed electric field applied in addition to the
baseline electric field is between 1 V/cm and 10 V/cm.
[0100] For pulsed electric fields which are symmetric square waves
the time the pulsed electric field is applied in addition to the
baseline electric field (ON) is the same as the time that the
pulsed electric field is not applied (OFF). In certain embodiments
the ON and OFF times are between 1 ms and 60 seconds. For pulsed
electric fields which are asymmetric square wave pulsed electric
fields have the time the pulsed electric field is applied in
addition to the baseline electric field (ON) is not the same as the
time that the pulsed electric field is not applied (OFF). In
certain embodiments the ON time is independently between 1 ms and
60 seconds, and the OFF time is independently between 1 ms and 60
seconds.
[0101] For pulsed electric fields which are symmetric triangular
waves the voltage ramp rate (V/s) up to the maximum electric field
applied is the same as the time that the voltage ramp rate (V/s)
down to the baseline electric field applied. In certain embodiments
the voltage ramp up and the voltage ramp down are between 10 mV/s
and 100 V/s. For pulsed electric fields which are asymmetric
triangular waves the voltage ramp rate (V/s) up to the maximum
electric field applied is not the same as the time that the voltage
ramp rate (V/s) down to the baseline electric field applied. In
certain embodiments the voltage ramp up is independently between 10
mV/s and 100 V/s and the voltage ramp down is independently between
10 mV/s and 100 V/s.
[0102] For pulsed electric fields which are symmetric sine waves
the period and frequency are constant, and the minimum electric
field of the sine wave is the same as the baseline electric field
applied. For pulsed electric fields which are asymmetric sine waves
the period and frequency are modulated, and the minimum electric
field of the sine wave is the same as the baseline electric field
applied.
[0103] The electrophoresis runs of the methods disclosed herein can
be performed at room temperature, ambient temperature, or at a
higher or lower temperature. By way of example only it may be
desirable for users to run their gels in a cold room with
pre-chilled buffers or at room temperature with pre-chilled
buffers. In certain embodiments the electrophoretic runs are
performed at lower temperatures include, but are not limited to,
temperatures from about 1.degree. C. to about 10.degree. C. For low
temperature runs, it can in some cases be preferable to run the gel
at a higher voltage at the end of the run.
[0104] The methods disclosed herein also include detecting one or
more bands on the electrophoresed gel that comprises a biomolecule.
One or more biomolecules or biomolecular complexes can be stained
or labeled before, during, or after electrophoresis using
techniques that are well known in the art. The bands can be
observed using light boxes, scanners, or by the naked eye without
special equipment. Optionally, the migration distance of one or
more bands of a sample can be determined. Optionally, molecular
weight markers can be electrophoresed on the same gel that the
sample is electrophoresed on, by applying a set of one or more
molecular weight markers on the gel to electrophoresis alongside
the one or more samples. The molecular weight of one or more bands
from the sample can be estimated or calculated by comparing the
migration of the band with that of one or more bands of the
molecular weight markers. The one or more bands can represent, for
example, proteins, protein complexes, nucleic acids, or nucleic
acid-protein complexes.
[0105] Visualization of the sample bands in the electrophoresis gel
can be achieved by illuminating the electrophoresis gel with light
of appropriate wavelength(s) to allow observation of dyes, stains
or other indicators associated with the sample bands. In certain
embodiments of the visualization methods, used in the methods
disclosed herein, the dyes, stains or other indicators are added to
the sample prior to loading in the electrophoresis gel. In other
embodiments, the dyes, stains or other indicators are added to the
loading well or loading wells prior to addition of the sample to
loading wells of the electrophoresis gel, while in other
embodiments the dyes, stains or other indicators are added to the
loading well or loading wells after to addition of the sample
loading wells of the electrophoresis gel. Alternatively, in certain
embodiments of the visualization methods, used in the methods
disclosed herein, the dyes, stains or other indicators are added to
the electrophoresis gel whereby they become associated with the
sample components during electrophoretic migration. In still other
embodiments of the visualization methods, used in the methods
disclosed herein, the dyes, stains or other indicators are
covalently attached to the sample components.
[0106] The systems, dyes and stains used for visualization can be
fluorescent or non-fluorescent. Non-limiting examples of the
systems, dyes and stains used in the methods disclosed herein are
silver staining or Coumassie Blue stain.
[0107] The light used for visualization can be monochromatic or
polychromatic. By way of example only, polychromatic light can be
white light, UV light or infra-red light, while monochromatic light
can be achieved using lasers or Light Emitting Diodes (LED's), or
by specific spectral filtering of sources such as white light, UV
light or infra-red light. It would be understood that the desired
wavelength of such monochromatic light depends on the specific
spectral characteristics of the dye or stain used, and the skilled
artisan will know the methods to obtain such monochromatic
light.
[0108] In certain embodiment visualization is performed in a stand
alone "light box" in which the electrophoresis cassette is placed
during or after electrophoretic separation of the sample. In such
light boxes the electrophoresis cassette can be illuminated from
above or below. Monitoring can be achieved using a CCD camera or a
video camera, or by direct observation of the user. In other
embodiments of such visualization methods an
electrophoresis/monitoring apparatus is used in which the
monitoring means (CCD camera or a video camera, or by direct
observation) and the means for application of the electric field or
fields are combined into one apparatus.
[0109] FIG. 1 compares electrophoretic separation of proteins and
protein complexes on a gel that includes linear acrylamide in the
stacker and a gel that does not include linear acrylamide in the
stacker. The electrophoresis gels used were 3-12% Blue Native
gradient gels without linear acrylamide in the stacking gel (top
gel) or with 0.05% linear acrylamide in the stacking gel (bottom
gel). Gels were run for 90 minutes at 150V using 50 mM BisTris, 50
mM Tricine running buffer (0.02% Coomassie G-250 in cathode buffer
only) and stained with colloidal Coomassie. Lanes 1, 5, and 10 were
loaded with 5 uL of unstained native standards (Invitrogen,
Carlsbad, Calif.); Lanes 2, 4, 7, and 9 were loaded with 4 uL of
bovine mitochondrial extract solubilized in 1% dodecylmaltoside.
Arrows indicate skirting artifacts (present in the top gel only).
The presence of linear acrylamide in the stacking gel reduced the
skirting effects as observed in the electrophoresis gel without the
linear acrylamide in the stacking gel.
EXAMPLES
Example 1
Native Protein/Protein Complex Gel Electrophoresis
[0110] Gel and Cassettes: 3-12% polyacrylamide gradient gels were
poured comprising acrylamide/bisacrylamide in a ratio of 25.5:1 in
BisTris-Cl, pH 7.0 and 0.3 mM CHAPS detergent. The stacker was 3%
acrylamide/bisacrylamide in a ratio of 25.5:1 and 0.05% w/v linear
acrylamide (600,000 to 1,000,000 Daltons). A first gel was poured
in a cassette in which the measurement of the gap in the middle of
the cassette along the upper edge was 0.923 mm, and a second gel
was poured in a cassette having a gap of 0.991 mm in the middle of
the cassette along the upper edge. The interplate gaps at the left
and right edges of these cassettes were between 1.01 mm and 1.02
mm, therefore the first gel cassette has a narrower middle region
compared with the second gel cassette.
[0111] Sample Preparation: Bovine mitochondria were isolated as
described previously (Rice, J. E. & Lindsay, J. G. (2002)
"Subcellular fractionation of mitochondria" in Subcellular
Fractionation: A Practical Approach, Edited by Graham, J. M. &
Rickwood D., Oxford University Press, New York, pp. 107-115).
Isolated mitochondria in TESS buffer (250 mM sucrose, 1 mM
succinate, 0.2 mM EDTA, 10 mM Tris pH 7.8 at 4.degree. C.) were
stored at -80.degree. C. in 250 uL aliquots. Aliquots were thawed
on ice before extracting mitochondrial proteins in cold 1.times.
Sample Buffer containing 1% dodecylmaltoside or 2% digitonin, 50 mM
BisTris-CL, pH, 7.0, 50 mM NaCl, 10% w/v glycerol and 0.001%
Ponceau S. Mitochondria were dissolved in buffer by pipetting up
and down through a yellow pipette tip and by inversion.
Mitochondrial extracts were incubated on ice for 15 minutes before
centrifuging 30 minutes at 20,000.times.g and 4.degree. C. Pellets
were discarded and supernatants were aliqotted and stored at
-80.degree. C. until used. Before loading onto the gel, 5% G-250
sample additive that included 5% Coomassie G-250 and 20.1% NDSB201
was added to the sample so that the final concentration of the
G-250 was one-fourth that of the detergent concentration in the
sample. The 5% G-250 sample additive was added to the samples while
they were on ice and just before loading onto the gel. For samples
that did not contain detergent, no 5% G-250 sample additive was
used.
[0112] Five microliters of unstained native protein standards
comprising IgM, Apoferritin, B-Phycoerythrin, Lactate
Dehydrogenase, Bovine Serum Albumin, and Soybean Trypsin Inhibitor
used as molecular weight markers on the gels.
[0113] Loading Samples onto NativePAGE.TM. Gels: Native gels were
prepared for sample application by removing the comb and tape then
rinsing the sample wells once with cathode buffer before again
filling the sample wells with cathode buffer. Samples were loaded
onto the gels either outside of the SureLock.TM. mini-cell
electrophoresis apparatus (Invitrogen Corp, Carlsbad Calif.) or in
the mini-cell before running buffer was added. Samples were loaded
by underlaying the sample beneath the cathode buffer by delivering
the sample to the bottom of the well in as thin a layer as
possible. An effort was also made to minimize the amount of time
between loading sample onto the gels and beginning
electrophoresis.
[0114] Running NativePAGE.TM. Gels: Running buffers were prepared
as shown in the following table. TABLE-US-00001 TABLE 1 Running
Buffers for Native Gel Electrophoresis Dark Blue Light Blue Anode
Cathode Cathode Component Buffer Buffer Buffer Running Buffer
(20.times.) 30 mL 10 mL 10 mL Cathode Additive 0 mL 10 mL 1 mL
(20.times.) Ultrapure water 570 mL 180 mL 189 mL
20.times. Running Buffer was 1 M BisTris, 1 M Tricine, pH 7.5-7.65.
Cathode additive was 0.4% Coomassie G-250 in water.
[0115] The type of cathode buffer used depended on the application
performed. Table 2 details the various cathode buffers used. By way
of example only, the dark blue cathode buffer contained 0.02% G-250
and was used in native electrophoresis runs where samples that
contain detergent were used, as shown in FIG. 3. A light blue
cathode buffer containing 0.002% G-250 and was used in native
electrophoresis runs where samples did not contain detergent, or
where the dark blue cathode buffer would interfere with downstream
applications. Downstream applications that benefit from the use of
the light blue cathode buffer are western blotting, silver
staining, and 2-dimensional native-SDS PAGE. For native
electrophoresis runs of detergent-containing samples intended for
downstream processing requiring light blue cathode buffer, the run
was started with the dark blue cathode buffer and after the
dye-migration front was one-third of the way down the gel the run
was paused, the dark blue cathode buffer removed with a serological
pipette and replaced by the light blue cathode buffer before
resuming the run. TABLE-US-00002 TABLE 2 Cathode Buffers for Native
Gel Electrophoresis Application: Detergent Non-detergent Detergent
Detergent Non-detergent samples, samples, samples, western samples,
samples, western Coomassie stain Coomassie stain blotting or 2D
Silver stain blotting or 2D Cathode Buffer: Dark Blue Dark Blue or
Dark Blue then Light Blue Light Blue Light Blue Light Blue
[0116] Electrophoresis was performed at 150V. The current limit was
set at 15 mA per gel. The run times for 3-12% gels were typically
90-100 minutes and the run times for 4-16% gels were typically
105-120 minutes when running at room temperature with room
temperature buffers.
[0117] Staining NativePAGE.TM. Gels: After running the native gels
they were deeply blue and some highly abundant protein bands were
visible; however, further staining of the gel was necessary. At the
end of the run, all proteins were still in their native folded
state, and in order to provide the most sensitive staining the
proteins were at least partially denatured to expose more
hydrophobic sites for dye-binding according to the follow
method.
[0118] Coomassie G-250 Long Protocol high sensitivity staining: The
gels were placed in 100 mL fix solution (40% methanol, 10% acetic
acid) and microwaved 45 seconds and shake for 15-30 minutes. The
fix step was repeated once for the 3-12% gels. The fix solution was
decanted, and 100 mL stain solution was added from the Colloidal
Blue Staining Kit (Invitrogen Corp., Carlsbad, Calif.; 55 mL water,
20 mL methanol, 20 mL stainer A, 5 mL stainer B) and the gels were
incubated overnight with shaking. Stain solution was decanted, and
100 mL 8% acetic acid was added. The gels were incubated with
shaking for 5 minutes (this removes any G-250 precipitated on the
surface of the gel or staining vessel). The acetic acid was then
decanted, and 100 mL distilled water was added and the gels were
shaken until desired background level was obtained.
[0119] The results, typified in FIG. 3, shows that the cassettes
with middle gap measurements that were similar to their upper gap
measurements produced non-skirting gels, while cassettes with
middle gap measurements significantly smaller than their upper gap
measurements produced skirting gels. Skirting artifacts in gel A
are indicated with arrows.
[0120] While the invention has been disclosed in connection with
illustrative embodiments, it is not intended to limit the scope of
the invention to the particular form set forth, but on the
contrary, it is intended to cover such alternatives, modifications,
and equivalents as may be included within the spirit and scope of
the invention as defined by the appended claims.
[0121] It should be understood that the foregoing description is
only illustrative of the invention. Headings are for convenience
only and are not intended to limited disclosure falling under a
heading to only that heading. Various alternatives and
modifications can be devised by those skilled in the art without
departing from the invention. Accordingly, the present invention is
intended to embrace all such alternatives, modifications and
variances which fall within the scope of the appended claims.
[0122] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference in their
entirety to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated by reference.
* * * * *