U.S. patent application number 12/862509 was filed with the patent office on 2011-03-31 for system for rapid high-resolution gel electrophoresis.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Jennifer Miller, Timothy UPDYKE.
Application Number | 20110073478 12/862509 |
Document ID | / |
Family ID | 43649893 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110073478 |
Kind Code |
A1 |
UPDYKE; Timothy ; et
al. |
March 31, 2011 |
SYSTEM FOR RAPID HIGH-RESOLUTION GEL ELECTROPHORESIS
Abstract
Electrophoretic systems, formulations and methods are described
which allow a user to perform electrophoresis experiments under
conditions of high voltage and with reduced run time. An
electrophoretic system, formulation or method may be run at 50%
higher field strength than comparable systems already in use in the
art. The presently described systems and formulations may be run at
voltages above 225 V, above 250 V, above 275 V, above 300 V, above
325 V or above 350 V. The time required for performing an
electrophoresis experiment may be reduced to less than about 30
minutes, less than about 20 minutes, less than about 15 minutes or
less than about 12 minutes.
Inventors: |
UPDYKE; Timothy; (Temecula,
CA) ; Miller; Jennifer; (Oceanside, CA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
43649893 |
Appl. No.: |
12/862509 |
Filed: |
August 24, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61236293 |
Aug 24, 2009 |
|
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|
Current U.S.
Class: |
204/616 ;
204/606 |
Current CPC
Class: |
G01N 33/561 20130101;
G01N 27/44747 20130101 |
Class at
Publication: |
204/616 ;
204/606 |
International
Class: |
G01N 27/447 20060101
G01N027/447 |
Claims
1. A discontinuous buffer electrophoretic system comprising: an
electrophoretic separation gel, said electrophoretic separation gel
comprising a gel buffer system, said buffer system comprising: a
gel amine buffer; and a gel ampholyte; and an aqueous running
buffer comprising a buffer ampholyte.
2. The discontinuous buffer electrophoretic system according to
claim 1, wherein the separation gel comprises acrylamide, agarose,
or acrylamide and agarose.
3. The discontinuous buffer electrophoretic system according to
claim 1, wherein the separation gel comprises acrylamide.
4. The discontinuous buffer electrophoretic system according to
claim 1, wherein the separation gel comprises between about 6 to
about 25 wt. % acrylamide.
5. The discontinuous buffer electrophoretic system according to
claim 1, wherein the separation gel comprises between about 8 to
about 20 wt. % acrylamide.
6. The discontinuous buffer electrophoretic system according to
claim 1, wherein the separation gel comprises between about 8 to
about 15 wt. % acrylamide.
7. The discontinuous buffer electrophoretic system according to
claim 1, wherein the electrophoretic separation gel further
comprises a stacking gel coupled to one end thereof.
8. The discontinuous buffer electrophoretic system according to
claim 7, wherein the stacking gel comprises about 4 wt. %
acrylamide.
9. The discontinuous buffer electrophoretic system according to
claim 1, wherein the gel ampholyte is different from the buffer
ampholyte.
10. The discontinuous buffer electrophoretic system according to
claim 1, wherein the pK of the gel amine buffer is in the range of
about 5.5 to about 7.5.
11. The discontinuous buffer electrophoretic system according to
claim 1, wherein the gel amine buffer is Bis-Tris.
12. The discontinuous buffer electrophoretic system according to
either claim 10 or claim 11, wherein the concentration of the gel
amine buffer is in the range of about 190 mM to about 295 mM.
13. The discontinuous buffer electrophoretic system according to
claim 1, wherein the concentration of the gel amine buffer is less
than about 300 mM.
14. The discontinuous buffer electrophoretic system according to
claim 1, wherein the concentration of the gel amine buffer is
between about 175 mM to about 300 mM.
15. The discontinuous buffer electrophoretic system according to
claim 1, wherein the pK of the gel ampholyte is about 1.5 pK units
greater than the pK of the gel amine.
16. The discontinuous buffer electrophoretic system according to
claim 1, wherein the pK of the gel ampholyte is between about 7 to
about 9.
17. The discontinuous buffer electrophoretic system according to
claim 1, wherein the pK of the gel ampholyte is between about 8 to
about 8.5.
18. The discontinuous buffer electrophoretic system according to
claim 1, wherein the pK of the gel ampholyte is between about 8.1
to about 8.3.
19. The discontinuous buffer electrophoretic system according to
claim 1, wherein the gel ampholyte is Tricine, Bicine, glycinamide
or acetamidoglycine.
20. The discontinuous buffer electrophoretic system according to
claim 1, wherein the gel ampholyte is Tricine or Bicine.
21. The discontinuous buffer electrophoretic system according to
either of claim 19 or 20, wherein the concentration of the gel
ampholyte is in the range of about 25 mM to about 125 mM.
22. The discontinuous buffer electrophoretic system according to
either of claim 19 or 20, wherein the concentration of the gel
ampholyte is in the range of about 50 mM to about 100 mM.
23. The discontinuous buffer electrophoretic system according to
either of claim 19 or 20, wherein the concentration of the gel
ampholyte is about 50 mM.
24. The discontinuous buffer electrophoretic system according to
claim 1, wherein the pH of the gel buffer system is in the range of
about 6 to about 8
25. The discontinuous buffer electrophoretic system according to
claim 1, wherein the pH of the gel buffer system is in the range of
about 6.0 to about 7.5.
26. The discontinuous buffer electrophoretic system according to
claim 1, wherein the pH of the gel buffer system is about 6.5.
27. The discontinuous buffer electrophoretic system according to
any of claims 24 to 26, wherein the pH is adjusted by the addition
of an acid.
28. The discontinuous buffer electrophoretic system according to
claim 27, wherein the acid is HCl.
29. The discontinuous buffer electrophoretic system according to
claim 1, wherein the gel buffer system further includes a source of
Cl.sup.- ions.
30. The discontinuous buffer electrophoretic system according to
claim 1, wherein the gel buffer system further includes about 95 mM
to about 170 mM Cl.sup.- ions.
31. The discontinuous buffer electrophoretic system according to
claim 1, wherein the gel buffer system further includes about 135
mM to about 145 mM Cl.sup.- ions.
32. The discontinuous buffer electrophoretic system according to
claim 1, wherein the pK of the buffer ampholyte in the running
buffer is less than the pK of the gel ampholyte.
33. The discontinuous buffer electrophoretic system according to
claim 1, wherein the buffer ampholyte comprises MES, ADA, PIPES,
ACES, BES, TES, HEPES, MOPS, CAPSO, DIPSO, POPSO, HEPPS, HEPPSO,
salts thereof, or any combination thereof.
34. The discontinuous buffer electrophoretic system according to
claim 1, wherein the buffer ampholyte comprises MES, MOPS or
DIPSO.
35. The discontinuous buffer electrophoretic system according to
claim 1, wherein the buffer ampholyte comprises MES.
36. The discontinuous buffer electrophoretic system according to
claim 1, wherein the buffer ampholyte comprises MOPS.
37. The discontinuous buffer electrophoretic system according to
claim 1, wherein the buffer ampholyte comprises DIPSO.
38. The discontinuous buffer electrophoretic system according to
claim 1, wherein the electrophoretic separation gel further
comprises sucrose.
39. The discontinuous buffer electrophoretic system according to
claim 38, wherein the concentration of sucrose is less than about 5
wt. %.
40. The discontinuous buffer electrophoretic system according to
claim 38, wherein the concentration of sucrose is less than about 3
wt. %.
41. The discontinuous buffer electrophoretic system according to
claim 38, wherein the concentration of sucrose is less than about 2
wt. %.
42. The discontinuous buffer electrophoretic system according to
claim 38, wherein the concentration of sucrose is less than about
1.5 wt. %.
43. The discontinuous buffer electrophoretic system according to
claim 1, further comprising SDS in at least the separating gel.
44. The discontinuous buffer electrophoretic system according to
claim 1, further comprising SDS in the separating gel and the
running buffer.
45. The discontinuous buffer electrophoretic system according to
claim 1, wherein the separating gel is a slab gel.
46. The discontinuous buffer electrophoretic system according to
claim 45, wherein the slab gel is between two glass or plastic
plates.
47. A kit comprising, in at least a first suitable container: one
or more gel cassettes, each of said gel cassettes comprising the
discontinuous buffer electrophoretic system according to any of
claims 1 to 47; and an aqueous running buffer according to any of
claims 1 to 47.
Description
BACKGROUND
[0001] 1. Field of the Invention
[0002] This invention relates to techniques and formulation for use
in gel electrophoresis. More particularly, the present invention
relates to novel systems and formulations for rapid, high
resolution gel electrophoresis at substantially neutral pH.
[0003] 2. Description of Related Art
[0004] Gel electrophoresis is a common procedure for the separation
of biological molecules, such as deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), polypeptides and proteins. In gel
electrophoresis, the molecules are separated into bands according
to the rate at which an imposed electric field causes them to
migrate through a filtering gel.
[0005] The basic apparatus used in this technique consists of a gel
enclosed in a glass tube or sandwiched as a slab between glass or
plastic plates. The gel has an open molecular network structure,
defining pores which are saturated with an electrically conductive
buffered solution of a salt. These pores through the gel are large
enough to admit passage of the migrating macromolecules.
[0006] The gel is placed in a chamber in contact with buffer
solutions which make electrical contact between the gel and the
cathode or anode of an electrical power supply. A sample containing
the macromolecules and a tracking dye is placed on top of the gel.
An electric potential is applied to the gel causing the sample
macromolecules and tracking dye to migrate toward the bottom of the
gel. The electrophoresis is halted just before the tracking dye
reaches the end of the gel. The locations of the bands of separated
macromolecules are then determined. By comparing the distance moved
by particular bands in comparison to the tracking dye and
macromolecules of known size, the size of other macromolecules can
be determined.
[0007] The rate of migration of macromolecules through the gel
depends upon four principle factors: the porosity of the gel; the
size and shape of the macromolecule; the charge density of the
macromolecule and the applied field strength. Electrophoresis
systems generally attempt to control these factors in order to be
reproducible from gel to gel and from sample to sample. However,
maintaining uniformity between gels is difficult because each of
these factors is sensitive to many variables in the chemistry of
the gel system.
[0008] Polyacrylamide gels are commonly used for electrophoresis.
Polyacrylamide gel electrophoresis or PAGE is popular because the
gels are optically transparent, electrically neutral and can be
made with a range of pore sizes. When used with sodium dodecyl
sulfate (SDS), i.e. as SDS-PAGE, the charge density of the
macromolecules is controlled by adding sodium dodecyl sulfate (SDS)
to the system. SDS molecules associate with the macromolecules and
impart a uniform charge density to them, substantially negating the
effects of any innate molecular charge.
[0009] Historically, SDS-PAGE gels were usually poured and run at
basic pH. The most common PAGE buffer system employed for the
separation of proteins is that developed by Ornstein and Davis
(Ornstein, L. (1964) Ann. NY Acad. Sci., 121: 321 and Davis, B. J.
(1964) Ann. NY Acad. Sci.: 121: 404), and modified for use with SDS
by Laemmli (Laemmli, 1970, Nature 227, 680-686). The Laemmli buffer
system consists of 0.375 M tris (hydroxymethyl) amino-methane
(Tris), titrated to pH 8.8 with HCl, in the separating gel. The
stacking gel consists of 0.125 M Tris, titrated to pH 6.8. The
anode and cathode running buffers contain 0.024 M Tris, 0.192 M
glycine, 0.1% SDS. An alternative buffer system is disclosed by
Schaegger and von Jagow (Schaegger, H. and von Jagow, G., Anal.
Biochem. 1987, 166, 368-379).
[0010] U.S. Pat. Nos. 7,422,670; 6,783,651; 6,143,154; 6,059,948;
6,096,182; 6,162,338; 5,922,185; and 5,578,180 by Updyke, et al.
describe gel and discontinuous buffer systems where separation
occurs at neutral pH and where the proteins remain in a
substantially reduced state. The gel systems include a gel amine
having a pK in the range of 5.5 to 7.5, and the pH of the gel
buffer is in the neutral range (pH 6-8). The gel system exhibits
improved stability of the gel matrix and stock solutions. The gels
can be stored under refrigeration for over a year without
substantial loss of performance. The gel system allows high
resolution of proteins that can cover separation range of 2 to 200
kDa. However, the resolution speed of the gel system could be
improved and the resolution of these gel formulations deteriorates
when field strength is increased by 50%.
[0011] There exists a need for electrophoresis gel formulations
that can run protein samples on SDS-PAGE gels with significantly
reduced run times while maintaining, or increasing, protein
resolution with pH neutral discontinuous buffer systems that
produce lower conductance while maintaining sufficient buffering
capacity to achieve such results using about 50% more field
strength than current electrophoresis gel formulations.
SUMMARY
[0012] In the presently described embodiments, electrophoretic
systems, formulations, kits and methods are described that allow a
user to perform electrophoresis experiments under conditions of
high voltage and reduced run time. The systems formulations and
methods improve upon those already known in the art and allow the
user to generate electrophoretically resolved protein samples with
a high degree of resolution and a reduced amount of time than
currently available systems and formulations. An electrophoretic
system, formulation or method practiced in accordance with the
presently disclosed embodiments may be run, for example, at 50%
higher field strength than comparable systems already in use in the
art. In some embodiments, an electrophoresis gel prepared in
accordance with the systems, methods and formulations described
herein may be run at voltages above 225 V, above 250 V, above 275
V, above 300 V, above 325 V or above 350 V. In some embodiments, an
electrophoresis gel prepared in accordance with the systems,
methods and formulations described herein may be using a field
strength in the range of about 12 v/cm to about 20 v/cm, or at
least 12.0-13.0 V/cm, at least 13.5-14 V/cm, at least 15.0-15.5
V/cm, at least 16.0-17.0 V/cm, or at least 17.5-18.5 V/cm.
[0013] In some embodiments, the time required for performing an
electrophoresis experiment may be reduced to less than about 30
minutes, less than about 20 minutes, less than about 15 minutes or
less than about 12 minutes.
[0014] In some embodiments, electrophoresis may be performed using
the gel formulations that form the basis of the present disclosure
using constant current.
[0015] In some embodiments, electrophoresis may be performed using
the gel formulations that form the basis of the present disclosure
using constant power.
[0016] An electrophoretic system and method in accordance with the
presently described embodiments may be part of a discontinuous
buffer system.
[0017] An electrophoretic system may include a polyacrylamide
electrophoretic separation gel. In some embodiments, a separation
gel may include a resolving gel portion and an optional stacking
gel portion cast over the resolving portion. In an embodiment, the
percentage of polyacrylamide, cross-linker, or both polyacrylamide
and cross-linker, present in the stacking portion may be different
from the percentage of polyacrylamide and/or cross-linker that is
present in the resolving portion.
[0018] In an embodiment, the resolving portion may include from
about 3% to about 25% polyacrylamide in combination with about 1%
to about 6% of a suitable cross-linker such as bis-acrylamide. In
an embodiment, the resolving gel portion may be polymerized using
from about 2% to about 5% cross-linker. In an embodiment, a
resolving gel may include up to about 25% polyacrylamide, up to
about 20% polyacrylamide, up to about 15% polyacrylamide, up to
about 12% polyacrylamide, up to about 10% polyacrylamide, up to
about 8% polyacrylamide, up to about 6% polyacrylamide, or up to
about 5% polyacrylamide.
[0019] In an embodiment, an electrophoretic separation gel may
optionally include, in the stacking portion, the resolving portion,
or both the stacking and resolving portions, a density agent such
as, e.g., sucrose, glycerol, or a similar non-ionic density agent.
The amount of a density agent included in the formulation may be in
the range of about 0 to about 10 vol. %, from about 0.5 to about 5
vol. %, from about 1.0 vol. % to about 3.5 vol. %, or about 1.5
vol. % to about 2 vol. %. In an embodiment, the concentration of
density present in a gel formulation may be up to about 10 vol. %,
up to about 8.5 vol. %, up to about 5 vol. %, up to about 4.5 vol.
%, up to about 4 vol. %, up to about 3.5 vol. %, up to about 3 vol.
%, up to about 2.5 vol. %, up to about 2 vol. %, up to about 1.5
vol., or up to about 1.0 vol. %.
[0020] In an embodiment, at least the resolving gel portion may
include a buffer system having a gel amine buffer in combination
with a gel ampholyte. In alternate embodiments, both the stacking
gel portion and the resolving gel portion may include a gel amine
buffer in combination with a gel ampholyte.
[0021] A variety of gel amine buffers may be used in the
preparation of the gel composition described herein. Gel amine
buffers particularly suited for use with the subject
electrophoretic separation gels and systems may include primary
organic amines or substituted amines. The pK.sub.a range of the gel
amine buffer may be near neutrality, typically in the range of
about 5 to about 8, in the range of about 5.5 to about 7.5, in the
range of about 6 to about 7, in the range of about 6.2 to about
6.8, or about 6.5. Exemplary though non-limiting gel amines buffers
suitable for use with the electrophoretic systems and methods
described herein may include
Bis(2-hydroxyethyl)-imino-tris(hydroxymethyl)-methane (hereinafter
"Bis-Tris"), 1,3-bis(tris(hydroxymethyl)methylamino)propane
(hereinafter "Bis-Tris propane"), tris(hydroxymethyl)aminomethane
(hereinafter "Tris"), triethaniolamine, or any derivatives or salts
thereof having a pKa value in the range of about 5.5 to about 7.5.
In some embodiments, the concentration of gel amine buffer present
in the gel may be in the range of about 150 mM to about 350 mM, or
in the range of about 165 mM to about 325 mM. Typically, the
concentration of gel amine buffer present in the gel may be less
than about 300 mM. In certain non-limiting embodiments, the
concentration of gel amine buffer present in a gel may be in the
range of about 175 mM to about 300 mM, or about 190 mM to about 295
mM. In some embodiments, the gel amine buffer selected may be
immobilized in the gel matrix after the polymerization of
acrylamide components to form the polyacrylamide matrix.
[0022] In some embodiments, gel ampholytes particularly suited for
use with the subject electrophoretic separation gels and systems
may include any biological buffer having an amine group and which
is capable of forming zwitterions in aqueous solution. In some
embodiments, gel ampholytes that are well-suited for use with the
subject gel formulation will have a pK value that is about 1.5 pH
units larger than the pKa value of the gel amine. By way of
non-limiting example, the following exemplary gel ampholytes are
contemplated for use with the presently described embodiments:
N-(Tri(hydroxymethyl)methyl)glycine (hereinafter "tricine"),
N,N-Bis(2-hydroxyethyl)glycine; Diethylolglycine (hereinafter
"bicine"), piperazine-N,N'-bis(2-ethanesulfonic acid) (hereinafter
"PIPES"), 3-(N-Morpholino)-2-hydroxypropanesulfonic Acid
(hereinafter "MOPSO"), N-(2-Acetamido)-2-aminoethanesulfonic acid
(hereinafter "ACES"), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic
acid (hereinafter "BES"),
N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (hereinafter
"TES"), -(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(hereinafter "HEPES"), 2-amino-methyl-1,3-propanediol
3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid
(hereinafter "HEPPS"), and
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid
(hereinafter "TAPS"). Of course, it will be readily apparent to
those skilled in the art that any gel ampholyte having at least one
amine or substituted amine and further having a pKa that is about
1.5 pH units greater than the gel amine chosen for use in the gel
may be used in the practice of the presently described embodiments,
without departing from the spirit and scope thereof. An ampholyte
may be present in a gel formulation at a concentration in the range
of about 25 mM to about 175 mM, about 45 mM to about 100 mM, about
50 mM to about 75 mM or about 50 mM to about 60 mM.
[0023] In some embodiments, the subject electrophoretic separation
gels and systems may be titrated with an appropriate acid such that
the desired pH of the electrophoresis gel is achieved. In an
embodiment, a sufficient amount of acid may be used such that the
pH of the electrophoresis gel is substantially neutral (i.e., in
the range of about pH 5.5 to about pH 7.5). In an embodiment, a
sufficient amount of acid may be used such that the pH of the
electrophoresis gel is in the range of about pH 5.5 to about pH
7.5, in the range of about pH 6 to about pH 7, or in the range of
about pH 6.2 to about pH 6.8. In an embodiment, a sufficient amount
of acid may be used to adjust the pH of the electrophoresis get to
substantially neutral, e.g., pH of about 6.2, about 6.3, about 6.4,
about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0,
about 7.1, or about 7.2. Typically, though not exclusively, the
acid chosen may include a source of anions, such as chloride ions,
phosphate ions, sulfate ions or acetate ions. The anions present in
the gel formulation may act, in part, as a source of leading ions
for a discontinuous gel system. An exemplary though non-limiting
acid suitable for use in titrating the gel buffer system of the
present embodiments is hydrochloric acid (HCl). In an embodiment,
an electrophoresis gel may be titrated with approximately half as
much HCl (on a molar basis) as the primary amine and/or the
ampholyte, so that the pH of the buffer is approximately neutral.
In some embodiments, the final concentration of chloride ions used
to titrate an electrophoresis gel may be in the range of about 95
mM to about 175 mM, from about 120 mM to about 160 mM, from about
135 mM to about 145 mM, or about 140 mM.
[0024] In an embodiment, an electrophoretic system may additionally
include an aqueous running buffer. The aqueous running buffer may
include a running buffer ampholyte. In an embodiment, the running
buffer may include an ampholyte that is different from the gel
ampholyte. In an embodiment, a running buffer ampholyte may include
at least one organic buffer ampholyte having at least one amine
group. In an embodiment, the pKa of a running buffer ampholyte that
is particularly suited for use with the presently described systems
and methods will be lower than the pKa of the gel buffer
ampholyte.
[0025] In some embodiments, an aqueous running buffer ampholyte
that is chosen for use with the subject electrophoresis systems may
form the basis of a discontinuous buffer system. In such a system,
the buffer ampholyte may function, in part, as a source of trailing
ions for a discontinuous buffer system.
[0026] Non-limiting examples of aqueous running buffer ampholytes
that are suitable for use with the presently described systems and
methods include 3-(N-morpholino) propanesulfonic acid ("MOPS"),
2-(N-morpholino) ethanesulfonic acid (MES),
3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic Acid
(hereinafter "DIPSO"), N'-bis(2-hydroxypropanesulfonic acid)
("POPSO"), HEPPS,
N-(2-Hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic Acid
("HEPPSO"), or
3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid
("TAPSO"), or any suitable salts thereof. Typically, the amount of
ampholyte present in an aqueous running buffer will be in the range
of about 10 mM to about 100 mM, about 15 mM to about 75 mM, about
25 mM to about 50 mM or about 40 mM to about 45 mM. The running
buffer may optionally include an additional buffering agent such as
Tris, in addition to SDS and/or EDTA. An exemplary though
non-limiting example of a running buffer well suited for use with
the presently disclosed embodiments may include 25-100 mM MES,
25-100 mM Tris, about 0.05 to about 0.5% SDS and about 0.5 mM to
about 2 mM EDTA.
[0027] In some embodiments, discontinuous buffer electrophoretic
systems prepared in accordance with the presently described systems
and methods may be run at voltages exceeding 250 V and achieve full
resolution of the sample in less than 30 minutes. In some
embodiments, an electrophoresis gel prepared in accordance with the
presently described systems and formulations may be run at voltages
above 225 V, above 250 V, above 275 V, above 300 V, above 325 V or
above 350 V. In some embodiments, the time required for performing
an electrophoresis experiment using the systems and methods and
gels described may be reduced to less than about 30 minutes, less
than about 20 minutes, less than about 15 minutes or less than
about 12 minutes.
[0028] In some embodiments, electrophoresis may be performed using
the gel formulations that form the basis of the present disclosure
using constant current. For example, in some embodiments, the
subject gel formulations may be subjected to electrophoresis using
a constant current up to about 200 mA, up to about 175 mA, up to
about 150 mA, up to about 125 mA, or up to about 115 mA.
[0029] Samples subjected to electrophoresis using the subject
electrophoretic separation systems and formulation under such
conditions may achieve substantially comparable resolution as gel
formulations currently in use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The above brief description as well as further objects,
features and advantages of the methods and apparatus of the present
invention will be more fully appreciated by reference to the
following detailed description of presently preferred but
nonetheless illustrative embodiments in accordance with the present
invention when taken in conjunction with the accompanying drawings
in which:
[0031] FIG. 1 shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 357 mM Bis-Tris, 210
mM Cl.sup.-, pH 6.5 is cast over an 8% resolving gel (S:R=1:4)
having 100 mM Bis-Tris, 75 mM Tricine, 4 wt. % sucrose and 20 mM
BES. 5 .mu.l of Mark12.TM. protein marker was resolved at 300 V for
15 min in MES SDS running buffer, and the gel was stained with
SimplyBlue.TM. SafeStain;
[0032] FIG. 2 shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 357 mM Bis-Tris, 210
mM Cl.sup.-, pH 6.5 is cast over an 8% resolving gel (S:R=41:59)
having 100 mM Bis-Tris, 75 mM Tricine, 4 wt. % sucrose and 20 mM
BES. 5 .mu.l of Mark12.TM. protein marker was resolved at 300 V for
16 min in MES SDS running buffer, and the gel was stained with
SimplyBlue.TM. SafeStain;
[0033] FIG. 3 shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 357 mM Bis-Tris, 150
mM H.sub.2SO.sub.4, pH 6.1 is cast over an 8% resolving gel
(S:R=41:59) having 100 mM Bis-Tris, 100 mM Tricine, and 4 wt. %
sucrose. 5 .mu.l of Mark12.TM. protein marker was resolved at 300 V
for 18 min in MES buffer, and the gel was stained with
SimplyBlue.TM. SafeStain;
[0034] FIG. 4A shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 178 mM Bis-Tris, 105
mM Cl.sup.-, pH 6.5 is cast over an 8% resolving gel (S:R=1:3)
having 178 mM Bis-Tris, 105 mM Cl.sup.-, no Tricine, and 4 wt. %
sucrose. 5 .mu.l Mark12.TM. protein marker was loaded in lanes 1-3,
5, 7, 9 and 10, 5 .mu.l SeeBlue Plus2.RTM. Pre-Stained Protein
Standard was loaded in lane 4, 10 .mu.g E. coli lysate was loaded
in lane 6, and 5 .mu.l Novex.RTM. Sharp Pre-Stained Protein
Standard was loaded in lane 8 and the gel was run in MES SDS
Running Buffer at 300 V for 13 minutes and stained with
SimplyBlue.TM. SafeStain;
[0035] FIG. 4B shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 178 mM Bis-Tris, 105
mM Cl.sup.-, pH 6.5 is cast over an 8% resolving gel (S:R=1:3)
having 178 mM Bis-Tris, 105 mM Cl.sup.-, no Tricine, and 4 wt. %
sucrose. 5 .mu.l Mark12.TM. protein marker was loaded in lanes 1-3,
5, 7, 9 and 10, 5 .mu.l SeeBlue Plus2.RTM. Pre-Stained Protein
Standard was loaded in lane 4, 10 .mu.g E. coli lysate was loaded
in lane 6, and 5 .mu.l Novex.RTM. Sharp Pre-Stained Protein
Standard was loaded in lane 8 and the gel was run in MES SDS
Running Buffer at 350 V for 10 minutes and stained with
SimplyBlue.TM. SafeStain;
[0036] FIG. 5 shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 228 mM Bis-Tris, 210
mM Cl.sup.-, 50 mM Tricine, 0% sucrose, pH 6.5 was cast over an 8%
resolving gel (S:R=1:4) having 228 mM Bis-Tris, 140 mM Cl.sup.-, 50
mM Tricine, and 4 wt. % sucrose and no BES. 5 .mu.l Mark12.TM.
protein marker was loaded in lanes 1, 2, 5-7, 9 and 10, 5 .mu.l
SeeBlue Plus2.RTM. Pre-Stained Protein Standard was loaded in lane
3, 10 .mu.g E. coli lysate was loaded in lane 4, and 5 .mu.l
Novex.RTM. Sharp Pre-Stained Protein Standard was loaded in lane 8,
the gel was run in MES SDS Running Buffer at 300 V for 13 minutes
and stained with SimplyBlue.TM. SafeStain;
[0037] FIG. 6A shows a representative NuPAGE.RTM. Novex 8% Bis-Tris
gel with 5 .mu.l Mark12.TM. protein marker loaded in lanes 1, 2,
5-6, 9 and 10, 5 .mu.l SeeBlue Plus2.RTM. Pre-Stained Protein
Standard loaded in lane 3, 10 .mu.g E. coli lysate loaded in lane
4, 10 .mu.l Mark12.TM. protein marker loaded in lane 7, and 5 .mu.l
NOVEX.RTM. Sharp Pre-Stained Protein Standard loaded in lane 8, run
in MES SDS Running Buffer at 200 V for 32 minutes and stained with
SimplyBlue.TM. SafeStain;
[0038] FIG. 6B shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 228 mM Bis-Tris, 140
mM Cl.sup.-, 50 mM Tricine, 0% sucrose, pH 6.5 is cast over an 8%
resolving gel (S:R=1:4) having 228 mM Bis-Tris, 140 mM Cl.sup.-, 50
mM Tricine, 4% sucrose, and no BES, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1, 2, 5-6, 9 and 10, 5 .mu.l
SeeBlue Plus2.RTM. Pre-Stained Protein Standard is loaded in lane
3, 10 .mu.g E. coli lysate is loaded in lane 4, 10 .mu.l Mark12.TM.
protein marker loaded in lane 7, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard is loaded in lane 8, and the gel is
run in MES SDS Running Buffer at 200 V for 29 minutes and stained
with SimplyBlue.TM. SafeStain;
[0039] FIG. 6C shows a representative NuPAGE.RTM. Novex 8% Bis-Tris
gel with 5 .mu.l Mark12.TM. protein marker loaded in lanes 1, 2,
5-6, 9 and 10, 5 .mu.l SeeBlue Plus2.RTM. Pre-Stained Protein
Standard loaded in lane 3, 10 .mu.g E. coli lysate loaded in lane
4, 10 .mu.l Mark12.TM. protein marker loaded in lane 7, and 5 .mu.l
Novex.RTM. Sharp Pre-Stained Protein Standard loaded in lane 8, run
in MES SDS Running Buffer at 250 V for 20 minutes and stained with
SimplyBlue.TM. SafeStain;
[0040] FIG. 6D shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 228 mM Bis-Tris, 140
mM Cl.sup.-, 50 mM Tricine, 0% sucrose, pH 6.5 is cast over an 8%
resolving gel (S:R=1:4) having 228 mM Bis-Tris, 140 mM Cl.sup.-, 50
mM Tricine, 4% sucrose, and no BES, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1, 2, 5-6, 9 and 10, 5 .mu.l
SeeBlue Plus2.RTM. Pre-Stained Protein Standard is loaded in lane
3, 10 .mu.g E. coli lysate is loaded in lane 4, 10 .mu.l Mark12.TM.
protein marker loaded in lane 7, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard is loaded in lane 8, and the gel is
run in MES SDS Running Buffer at 250 V for 19 minutes and stained
with SimplyBlue.TM. SafeStain;
[0041] FIG. 6E shows a representative NuPAGE.RTM. Novex 8% Bis-Tris
gel with 5 .mu.l Mark12.TM. protein marker loaded in lanes 1, 2,
5-6, 9 and 10, 5 .mu.l SeeBlue Plus2.RTM. Pre-Stained Protein
Standard loaded in lane 3, 10 .mu.g E. coli lysate loaded in lane
4, 10 .mu.l Mark12.TM. protein marker loaded in lane 7, and 5 .mu.l
Novex.RTM. Sharp Pre-Stained Protein Standard loaded in lane 8, run
in MES SDS Running Buffer at 300 V for 16 minutes and stained with
SimplyBlue.TM. SafeStain;
[0042] FIG. 6F shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 228 mM Bis-Tris, 140
mM Cl.sup.-, 50 mM Tricine, 0% sucrose, pH 6.5 is cast over an 8%
resolving gel (S:R=1:4) having 228 mM Bis-Tris, 140 mM Cl.sup.-, 50
mM Tricine, 4% sucrose, and no BES, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1, 2, 5-6, 9 and 10, 5 .mu.l
SeeBlue Plus2.RTM. Pre-Stained Protein Standard is loaded in lane
3, 10 .mu.g E. coli lysate is loaded in lane 4, 10 .mu.l Mark12.TM.
protein marker loaded in lane 7, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard is loaded in lane 8, and the gel is
run in MES SDS Running Buffer at 300 V for 16 minutes and stained
with SimplyBlue.TM. SafeStain;
[0043] FIG. 7A shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 228 mM Bis-Tris, 140
mM Cl.sup.-, 50 mM Tricine, 0% sucrose, pH 6.5 was cast over an 8%
resolving gel (S:R=1:4) having 228 mM Bis-Tris, 140 mM Cl.sup.-, 50
mM Tricine, 1.5% sucrose, and no BES, where 5 .mu.l Mark12.TM.
protein marker was loaded in lanes 1, 2, 4-7, 9 and 10, 10 .mu.g E.
coli lysate was loaded in lane 3, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard was loaded in lane 8, the gel was run
at 300 V for 14 min in MES SDS running buffer and the gel was
stained with SimplyBlue.TM. SafeStain;
[0044] FIG. 7B shows a representative image of a 20 well
NuPAGE.RTM. Novex 8% Bis-Tris gel, where lanes 1, 3, 8, 10, 11, 13,
18 and 19 were loaded with 5 .mu.l Mark12.TM. protein marker, lane
5 was loaded with 10 .mu.g E. coli lysate and lane 16 was loaded
with 10 .mu.l Novex.RTM. Sharp Pre-Stained Protein Standard, the
gels was run at 200 V for 35 minutes in MES buffer and stained with
SimplyBlue.TM. SafeStain;
[0045] FIG. 7C shows a representative image of a 20 well
NuPAGE.RTM. Novex 8% Bis-Tris gel, where lanes 1, 3, 8, 10, 11, 13,
18 and 19 were loaded with 5 .mu.l Mark12.TM. protein marker, lane
5 was loaded with 10 .mu.g E. coli lysate and lane 16 was loaded
with 10 .mu.l Novex.RTM. Sharp Pre-Stained Protein Standard, the
gels was run at 250 V for 22 minutes in MES buffer and stained with
SimplyBlue.TM. SafeStain;
[0046] FIG. 7D is a bar graph indicating the relative sharpness of
each individual band (measured by pixel area) of the Mark12.TM.
protein marker set that was averaged over 7 independent experiments
using the gel formulation described in FIG. 7A (grey bars), FIG. 7B
(dark bars) and FIG. 7C (white bars). The X-axis indicates the band
number, with band number 1 being the 200 kDa Mark12.TM. band and
band 12 being the 2.5 kDa band;
[0047] FIG. 7E is a graph depicting the average R.sub.f value
obtained for each gel formulation described in FIG. 7A, FIG. 7B and
FIG. 7C using either NonLinear Dynamics TOTALLAB.TM. v2003.02
software or QC Software as indicated;
[0048] FIG. 8A shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 228 mM Bis-Tris, 140
mM Cl.sup.-, 50 mM Tricine, 0% sucrose, pH 6.5 is cast over a 10%
resolving gel (S:R=1:4) having 228 mM Bis-Tris, 140 mM Cl.sup.-, 50
mM Tricine, 1.75% sucrose, and no BES, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1, 2, 4-7, 9 and 10, 10 .mu.g E.
coli lysate is loaded in lane 3, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard is loaded in lane 8, the gel was run
at 300 V for 16 min in MES SDS running buffer and the gel was
stained with SimplyBlue.TM. SafeStain;
[0049] FIG. 8B shows a representative image of a 10 well
NuPAGE.RTM. Novex 10% Bis-Tris gel, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1, 2, 4-7, 9 and 10, 10 .mu.g E.
coli lysate is loaded in lane 3, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard is loaded in lane 8, the gels was run
at 200 V for 36 minutes in MES buffer and stained with
SimplyBlue.TM. SafeStain;
[0050] FIG. 8C shows a representative image of a 10 well
NuPAGE.RTM. Novex 10% Bis-Tris gel, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1, 2, 4-7, 9 and 10, 10 .mu.g E.
coli lysate is loaded in lane 3, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard is loaded in lane 8, the gels was run
at 250 V for 24 minutes in MES buffer and stained with
SimplyBlue.TM. SafeStain;
[0051] FIG. 8D is a bar graph indicating the relative sharpness of
each individual band (measured by pixel area) of the Mark12.TM.
protein marker set that was averaged over 7 independent experiments
using the gel formulation described in FIG. 8A (grey bars), FIG. 8B
(dark bars) and FIG. 8C (white bars). The X-axis indicates the band
number, with band number 1 being the 200 kDa Mark12.TM. band and
band 12 being the 2.5 kDa band;
[0052] FIG. 8E is a graph depicting the average R.sub.f value
obtained for each gel formulation described in FIG. 8A, FIG. 8B and
FIG. 8C using either TOTALLAB.TM. software or QC Software as
indicated;
[0053] FIG. 9A shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 228 mM Bis-Tris, 140
mM Cl.sup.-, 50 mM Tricine, 0% sucrose, pH 6.5 is cast over a 4-12%
gradient gel having 228 mM Bis-Tris, 140 mM Cl.sup.-, 50 mM
Tricine, 1.5-2% sucrose, and no BES, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1, 2, 4-7, 9 and 10, 10 .mu.g E.
coli lysate is loaded in lane 3, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard is loaded in lane 8, the gel was run
at 300 V for 17 min in MES SDS running buffer and the gel was
stained with SimplyBlue.TM. SafeStain;
[0054] FIG. 9B shows a representative image of a 10 well
NuPAGE.RTM. Novex 4-12% Bis-Tris gel, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1, 2, 4-7, 9 and 10, 10 .mu.g E.
coli lysate is loaded in lane 3, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard is loaded in lane 8, the gels was run
at 200 V for 37 minutes in MES buffer and stained with
SimplyBlue.TM. SafeStain;
[0055] FIG. 9C shows a representative image of a 10 well
NuPAGE.RTM. Novex 4-12% Bis-Tris gel, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1, 2, 4-7, 9 and 10, 10 .mu.g E.
coli lysate is loaded in lane 3, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard is loaded in lane 8, the gels was run
at 250 V for 25 minutes in MES buffer and stained with
SimplyBlue.TM. SafeStain;
[0056] FIG. 9D is a bar graph indicating the relative sharpness of
each individual band (measured by pixel area) of the Mark12.TM.
protein marker set that was averaged over 7 independent experiments
using the gel formulation described in FIG. 9A (grey bars), FIG. 9B
(dark bars) and FIG. 9C (white bars). The X-axis indicates the band
number, with band number 1 being the 200 kDa Mark12.TM. band and
band 12 being the 2.5 kDa band;
[0057] FIG. 9E is a graph depicting the average R.sub.f value
obtained for each gel formulation described in FIG. 9A, FIG. 9B and
FIG. 9C using either TOTALLAB.TM. software or QC Software as
indicated;
[0058] FIG. 10A shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 357 mM Bis-Tris, 210
mM Cl.sup.-, pH 6.5 is cast over a 12% resolving gel (S:R=1:4)
having 228 mM Bis-Tris, 140 mM Cl.sup.-, 50 mM Tricine, 2% wt. %
sucrose, and no BES, where 5 .mu.l Mark12.TM. protein marker is
loaded in lanes 1-10. The gel was run at 300 V for 15 min in MES
SDS running buffer and the gel was stained with SimplyBlue.TM.
SafeStain;
[0059] FIG. 10B shows a representative test gel according to one
embodiment, in which a 4% stacking gel having 228 mM Bis-Tris, 140
mM Cl.sup.-, 50 mM Tricine, 0% sucrose, pH 6.5 is cast over a 12%
resolving gel (S:R=1:4) having 228 mM Bis-Tris, 140 mM Cl.sup.-, 50
mM Tricine, 4% wt. % sucrose, and no BES, where 5 .mu.l Mark12.TM.
protein marker is loaded in lanes 1-10. The gel was run at 300 V
for 26 min in MES SDS running buffer and the gel was stained with
SimplyBlue.TM. SafeStain;
[0060] FIG. 10C is a bar graph indicating the relative sharpness of
each individual band (measured by pixel area) of the Mark12.TM.
protein marker set that was averaged over four independent
experiments using the gel formulation described in FIG. 10A (black
bars) and FIG. 10B (hatched bars) The X-axis indicates the band
number, with band number 1 being the 200 kDa Mark12.TM. band and
band 12 being the 2.5 kDa band.
[0061] While the invention is susceptible to various modifications
and alternative forms, specific embodiments thereof are shown by
way of example in the drawings and will herein be described in
detail. The drawings may not be to scale. It should be understood
that the drawings and detailed description thereto are not intended
to limit the invention to the particular form disclosed, but to the
contrary, the intention is to cover all modifications, equivalents
and alternatives falling within the spirit and scope of the present
invention as defined by the appended claims.
DETAILED DESCRIPTION OF EMBODIMENTS
Definitions
[0062] The terms used throughout this specification generally have
their ordinary meanings in the art, within the context of the
invention, and in the specific context where each term is used.
Certain terms are discussed below, or elsewhere in the
specification, to provide additional guidance to the practitioner
in describing the devices and methods of the invention and how to
make and use them. It will be appreciated that the same thing can
be said in more than one way. Consequently, alternative language
and synonyms may be used for any one or more of the terms discussed
herein, nor is any special significance to be placed upon whether
or not a term is elaborated or discussed in greater detail herein.
Synonyms for certain terms are provided. A recital of one or more
synonyms does not preclude the use of other synonyms. The use of
examples anywhere in this specification, including examples of any
terms discussed herein, is illustrative only, and in no way limits
the scope and meaning of any of the embodiments set forth herein or
of any exemplified term.
[0063] As used herein, the term "discontinuous buffer",
"discontinuous system" and "discontinuous buffer system" generally
refer to one of two aqueous buffering systems used in
polyacrylamide gel electrophoresis systems. A discontinuous buffer
system is functionally and chemically distinguished from a
continuous buffer system. Typically, a continuous system has only a
single separating gel and uses the same buffer in the tanks and the
gel. In a discontinuous system, a non-restrictive large pore gel,
called a stacking gel, is layered on top of a separating gel
(sometimes referred to as a "resolving gel"). Each gel may be made
with the same, or different, buffer, and the tank buffers are
different from the gel buffers. The discontinuity is created by the
difference in ion mobility of the faster moving leading ion (for
example, chloride) and the slower moving trailing ion (MES, MOPS,
glycine or tricine). The resolution obtained in a discontinuous
system is much greater than that obtained with a continuous system
for samples that contain dilute solutions of analytes due to the
concentrating effect of discontinuous systems to create sharp
starting zones of analyte molecules. The difference between a
continuous buffer system and a discontinuous buffer system is well
known to those skilled in the art to which the present invention
pertains.
[0064] The term "band sharpness" is a relative term that is used to
compare the sharpness of migrated bands as they appear on various
gel formulations. The term is used particularly when comparing
relative band sharpness between two or more of various gel
formulations. A variety of methods may be used to determine band
sharpness. For example, depending on the degree of resolution that
is obtained, band sharpness, or the degree to which band sharpness
is improved, may be determined by visual inspection. To obtain a
more quantitative estimation of band sharpness, a user may employ
gel imaging software. An exemplary though non-limiting gel analysis
software typically used in the art is, e.g., TOTALLAB.TM. software
(Nonlinear Dynamics Ltd, Durham, N.C.), GEL-PRO.TM. Analyzer (Media
Cybernetics, Bethesda, Md.) or LABIMAGE.RTM. 1D L300 Gel Analysis
software (LabImage, Leipzig, Germany). Band sharpness may be
determined by measuring the area of each resolved band in a gel,
and dividing the area by the width of each well (which is generally
constant). Another method of determining band sharpness may be to
directly measure the width of individual peaks detected by the gel
analysis algorithm.
[0065] Electrophoretic systems, formulations and methods are
described herein that allow a user to perform electrophoresis
experiments under conditions of high voltage and reduced run time.
The systems formulations and methods improve upon those already
known in the art and allow the user to generate electrophoretically
resolved proteins with a high degree of resolution and a reduced
amount of run time than currently available systems and
formulations. An electrophoretic system, formulation or method
practiced in accordance with the presently disclosed embodiments
may be run at 50% higher field strength than comparable systems
already in use in the art. By way of example, an electrophoresis
gel prepared in accordance with the presently described systems and
formulations may be run at voltages above 225 V, above 250 V, above
275 V, above 300 V, above 325 V or above 350 V. The time required
for performing the electrophoresis may be reduced to less than
about 30 minutes, less than about 20 minutes, less than about 15
minutes or less than about 12 minutes. In some embodiments,
electrophoresis may be performed using the gel formulation that
forms the basis of the present disclosure using constant current.
For example, in some embodiments, the subject gel formulations may
be subjected to electrophoresis using a constant current of up to
about 200 mA, up to about 175 mM, up to about 150 mM, up to about
125 mM, or up to about 115 mM.
[0066] In an embodiment, an electrophoretic system may include a
gel and buffer system wherein separation occurs at substantially
neutral pH. Without being bound by any particular theory or
mechanism, it is believe that the use of a neutral pH
electrophoretic system ensures that biological molecules within a
sample being subjected to electrophoresis (e.g., proteins, nucleic
acids, carbohydrates and the like) remain completely or
substantially reduced and unhydrolyzed. Advantageously, at neutral
pH, primary amino groups of proteins are less reactive with
unpolymerized acrylamide, thereby allowing greater resolution and
improved band sharpness. Additionally, at neutral pH, thiol groups
are less susceptible to oxidation than under the more alkaline
conditions under which SDS-PAGE is traditionally performed. The gel
formulations that form the basis of the present disclosure may, in
some embodiments, function optimally when used as part of a
discontinuous buffer system. A discontinuous buffer system is one
using a different aqueous buffer in the anode chamber, the cathode
chamber, or both the anode and the cathode chambers from the buffer
present in the gel. The concentration of the aqueous running buffer
may be different from the buffer concentration of the
electrophoresis gel. In some embodiments, a gel formulation in
accordance with the present disclosure may include a stacking gel
and a resolving gel.
[0067] The resulting gel electrophoresis systems display improved
stability of the gel matrix and buffer solutions at high voltages,
and thereby allow gel electrophoresis to be performed at higher
voltage than traditional electrophoretic systems, without
compromising band resolution. Gels prepared in accordance with the
presently described formulations and methods can be stored under
refrigeration for at least up to about sixteen months without loss
of performance. Additionally, stock buffers and stock gel solutions
without polymerization initiator can be stored for several weeks or
more at room temperature, with no loss of performance. Using the
systems and methods described herein, an 8% polyacrylamide gel, for
example, can resolve a protein sample in a separation range of
about 2 to 250 kDa in less than 15 minutes.
[0068] In some embodiments, a gel formulation may include a
stacking gel and a resolving gel. The ratio of stacking gel to
resolving gel (also denoted as S:R) may be about 0.5:9.5, about
1:9, about 1.5:8.5, about 2:8, about 2.5:7.5, about 3:7, about
3.5:6.5, about 4:6, about 4.5:5.5, or about 1:1. The meaning of the
term stacking and resolving gel, when used in the context of a
discontinuous gel electrophoresis system, is well known to a
practitioner having ordinary skill in the art. Typically, the
percentage of polyacrylamide and/or cross linker (for example,
bis-acrylamide) that is present in a stacking gel may be less than
the percentage of polyacrylamide and/or cross linker that is
present in a resolving gel. In some embodiments, the percentage of
polyacrylamide that is present in a stacking gel will be less than
about 6%, less than about 5%, less than about 4%, less than about
3%.
[0069] In one embodiment of an electrophoretic system and
formulation, a resolving polyacrylamide gel of between about 3% and
about 25% (% T) acrylamide is polymerized using about 1% to about
6% of a suitable crosslinker (% C) such as bisacrylamide. In an
embodiment, the gel is polymerized using from about 2% to about 5%
crosslinker (% C) in the presence of a gel buffer. In an
embodiment, a polyacrylamide gel may include 25% acrylamide, 24%
acrylamide, 23% acrylamide, 22% acrylamide, 21% acrylamide, 20%
acrylamide, 19% acrylamide, 18% acrylamide, 17% acrylamide, 16%
acrylamide, 15% acrylamide, 14% acrylamide, 13% acrylamide, 12%
acrylamide, 11% acrylamide, 10% acrylamide, 9% acrylamide, 8%
acrylamide, 7% acrylamide, 6% acrylamide, or 5% acrylamide.
[0070] In an embodiment of an electrophoretic system and
formulation, a reducing agent may optionally be included in one or
more of the gel, the buffer or the loading buffer, so as to
maintain a chemically reducing environment. A variety of suitable
reducing agents are known in the art, any of which are suitable for
use with the embodiments disclosed herein. By way of non-limiting
example, a reducing agent such as thioglycolic acid (TGA) or sodium
bisulfite may be added to the formulation. Suitable concentration
ranges for such reducing agents may be from about 0.5 mM to about
20 mM, from about 1 mM to about 15 mM, from about 2 mM to about 10
mM or from about 5 mM to about 7.5 mM. In some embodiments that
include an optional reducing agent, the reducing agent may be added
to the gel running buffer.
[0071] In certain non-limiting though preferred embodiments, the
SDS may be substantially absent from the electrophoresis gel and
may instead by provided in the running buffer and/or the loading
buffer. A variety of suitable running buffers are described in
detail below.
[0072] In some embodiments, a polyacrylamide gel in accordance with
the presently described embodiments may include primary organic
amine or mono- or di-substituted amine buffers. The pK.sub.a range
of the gel amine buffer can be near neutrality, typically in the
range of about 5 to about 8, in the range of about 5.5 to about
7.5, in the range of about 6 to about 7, in the range of about 6.2
to about 6.8, or about 6.5. The pK.sub.a range of the gel amine
buffer may be near neutrality, typically in the range of about 5 to
about 8, in the range of about 5.5 to about 7.5, in the range of
about 6 to about 7, in the range of about 6.2 to about 6.8, or
about 6.5. Exemplary though non-limiting gel amines buffers
suitable for use with the electrophoretic systems and methods
described herein may include
Bis(2-hydroxyethyl)-imino-tris(hydroxymethyl)-methane (hereinafter
"Bis-Tris"), 1,3-bis(tris(hydroxymethyl)methylamino)propane
(hereinafter "Bis-Tris propane"), tris(hydroxymethyl)aminomethane
(hereinafter "Tris"), triethaniolamine, or any derivatives or salts
thereof having a pKa value in the range of about 5.5 to about 7.5.
Of course, it will be readily apparent to those skilled in the art
that any gel amine buffer having at least one primary amine or
substituted amine and further having a pKa in the range of 5.5 to
7.5 may be used during the practice of the presently described
embodiments, without departing from the spirit and scope
thereof.
[0073] In some embodiments, the concentration of gel amine buffer
present in the gel may be in the range of about 150 mM to about 350
mM, or in the range of about 165 mM to about 325 mM. Typically, the
concentration of gel amine buffer present in the gel may be less
than about 300 mM. In certain non-limiting embodiments, the
concentration of gel amine buffer present in a gel may be in the
range of about 175 mM to about 300 mM, or about 190 mM to about 295
mM.
[0074] In some embodiments, the gel amine buffer may be added
directly to the resolving gel, to the stacking gel, or to both the
resolving gel and the stacking gel as an aqueous solution, at the
time the other aqueous components thereof are combined and prior to
the initiation of the polymerization reaction. Alternatively, the
gel amine buffer may be incorporated into the gel matrix by soaking
the polymerized gel in an aqueous solution containing the desired
concentration of gel amine buffer and allowing the gel amine buffer
to permeate the polymerized gel matrix.
[0075] In some embodiments, a polyacrylamide gel that forms the
basis of an electrophoretic system may include a gel ampholyte in
combination with the gel amine described above. A gel ampholyte
suitable for use with the subject gel formulations may include,
without limitation, any biological buffer having an amine group and
which is capable of forming zwitterions in aqueous solution. In
some embodiments, gel ampholytes that are well-suited for use with
the subject gel formulation will have a pK value that is about 1.5
pH units larger than the pKa value of the gel amine. By way of
non-limiting example, the following exemplary gel ampholytes are
contemplated for use with the presently described embodiments:
N-(Tri(hydroxymethyl)methyl)glycine (hereinafter "tricine"),
N,N-Bis(2-hydroxyethyl)glycine; Diethylolglycine (hereinafter
"bicine"), piperazine-N,N'-bis(2-ethanesulfonic acid) (hereinafter
"PIPES"), 3-(N-Morpholino)-2-hydroxypropanesulfonic Acid
(hereinafter "MOPSO"), N-(2-Acetamido)-2-aminoethanesulfonic acid
(hereinafter "ACES"), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic
acid (hereinafter "BES"),
N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (hereinafter
"TES"), -(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(hereinafter "HEPES"), 2-amino-methyl-1,3-propanediol
3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid
(hereinafter "HEPPS"), and
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid
(hereinafter "TAPS"). Of course, it will be readily apparent to
those skilled in the art that any gel ampholyte having at least one
amine or substituted amine and further having a pKa that is about
1.5 pH units greater than the gel amine chosen for use in the gel
may be used in the practice of the presently described embodiments,
without departing from the spirit and scope thereof. An ampholyte
may be present in a gel formulation at a concentration in the range
of about 25 mM to about 175 mM, about 45 mM to about 100 mM, about
50 mM to about 75 mM or about 50 mM to about 60 mM.
[0076] In some embodiments, the gel ampholyte may be added directly
to the resolving gel, to the stacking gel, or to both the resolving
gel and the stacking gel as an aqueous solution, at the time the
other aqueous components thereof are combined and prior to the
initiation of the polymerization reaction. Alternatively, the gel
ampholyte may be incorporated into the gel matrix by soaking the
polymerized gel in an aqueous solution containing the desired
concentration of gel ampholyte and allowing the gel ampholyte to
permeate the polymerized gel matrix.
[0077] In some embodiments, the subject buffered gel systems and
formulations may be titrated with an appropriate acid such that the
desired pH of the electrophoresis gel is achieved. In an
embodiment, a sufficient amount of acid may be used such that the
pH of the electrophoresis gel is substantially neutral (i.e., in
the range of about pH 5.5 to about pH 7.5). In an embodiment, a
sufficient amount of acid may be used such that the pH of the
electrophoresis gel is in the range of about pH 5.5 to about pH
7.5, in the range of about pH 6 to about pH 7, or in the range of
about pH 6.2 to about pH 6.8. In an embodiment, a sufficient amount
of acid may be used to adjust the pH of the electrophoresis get to
substantially neutral, e.g., pH of about 6.2, about 6.3, about 6.4,
about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0,
about 7.1, or about 7.2. Typically, though not exclusively, the
acid chosen may include a source of anions, such as chloride ions,
phosphate ions, sulfate ions or acetate ions. The anions present in
the gel formulation may act, in part, as a source of leading ions
for a discontinuous gel system. An exemplary though non-limiting
acid suitable for use in titrating the gel buffer system of the
present embodiments is hydrochloric acid (HCl). In an embodiment,
an electrophoresis gel may be titrated with approximately half as
much HCl (on a molar basis) as the primary amine and/or the
ampholyte, so that the pH of the buffer is approximately neutral.
In some embodiments, the final concentration of chloride ions used
to titrate an electrophoresis gel may be in the range of about 95
mM to about 175 mM, from about 120 mM to about 160 mM, from about
135 mM to about 145 mM, or about 140 mM.
[0078] In some embodiments, anions that function as a leading ion
in a discontinuous buffer system may be present in the stacking
gel, in the resolving gel, or in both the stacking gel and the
resolving. Exemplary though non-limiting leading ions suitable for
use in the presently described gel formulation may include anions,
such as chloride, phosphate ions, sulfate ions or acetate ions.
Sources for such anions may include HCl or any acid halide,
phosphoric acid, sulfuric acid, or acetic acid.
[0079] In an embodiment, the concentration of leading ion may be
higher in the stacking gel than in the resolving gel. In another
embodiment, the concentration of leading ion may be substantially
the same in the stacking gel as in the resolving gel. In some
embodiments, the concentration of leading ion in the stacking gel
may be up to about 350 mM, up to about 300 mM, up to about 250 mM,
up to about 200 mM, up to about 150 mM, up to about 125 mM, up to
about 110 mM, up to about 100 mM or up to about 75 mM.
[0080] In some embodiments, an electrophoresis gel in accordance
with the presently described embodiments may optionally include a
density agent such as, e.g., sucrose, glycerol, or a similar agent.
The amount of a density agent included in the formulation may be in
the range of about 0 to about 8 wt. %, form about 0.5 to about 5
wt. %, from about 1.0 wt. % to about 3.5 wt. %, or about 1.5 wt. %
to about 2 wt. %. In an embodiment, the concentration of sucrose
present in a gel formulation may be up to about 10%, up to about
8.5%, up to about 5%, up to about 4.5%, up to about 4%, up to about
3.5%, up to about 3%, up to about 2.5%, up to about 2%, up to about
1.5%, up to about 1% or up to about 0.5%. In some embodiments, the
amount of density agent present in an electrophoresis gel may vary.
Such embodiments may advantageously be used during the preparation
of a gradient gel, such as the methods described in, for example,
U.S. Pat. Nos. 6,472,503 and 6,197,173.
[0081] The gels prepared in accordance with the formulations
disclosed above may be subjected to electrophoresis using an
aqueous running buffer system. Typically, the aqueous running
buffer system may include at least one buffer ampholyte having at
least one amine group. In an embodiment, the running buffer
ampholyte that is selected for use with the subject discontinuous
electrophoresis system will be different from the gel buffer
ampholyte that is present in the electrophoresis gel being used
with the aqueous running buffer. In an embodiment, the pK.sub.a of
the selected running buffer ampholyte may be less than the pK.sub.a
of the gel buffer ampholyte being used.
[0082] In certain particularly preferred embodiments, the aqueous
running buffer ampholyte that is chosen will preferably form the
basis of a discontinuous buffer system. In such a system, the
running buffer ampholyte may function, in part, as a source of
trailing ions for a discontinuous buffer system. In an embodiment,
the pKa of a running buffer ampholyte that is particularly suited
for use with the presently described systems and methods will be
lower than the pKa of the gel buffer ampholyte.
[0083] Non-limiting examples of aqueous running buffer ampholytes
that are suitable for use with the presently described systems and
methods include 3-(N-morpholino) propanesulfonic acid ("MOPS"),
2-(N-morpholino) ethanesulfonic acid (MES),
3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic Acid
(hereinafter "DIPSO"), N'-bis(2-hydroxypropanesulfonic acid)
("POPSO"), HEPPS,
N-(2-Hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic Acid
("HEPPSO"), or
3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid
("TAPSO"), or any suitable salts thereof. Typically, the amount of
ampholyte present in an aqueous running buffer will be in the range
of about 10 mM to about 100 mM, about 15 mM to about 75 mM, about
25 mM to about 50 mM or about 40 mM to about 45 mM. The running
buffer may optionally include an additional buffering agent such as
tris, in addition to SDS and EDTA. An exemplary though non-limiting
example of a running buffer well suited for use with the presently
disclosed embodiments may include 25-100 mM MES, 25-100 mM Tris,
about 0.05 to about 0.5% SDS and about 0.5 mM to about 2 mM
EDTA.
[0084] This gel and buffer system also possesses improved stability
of the gel matrix and stock solutions. Gels prepared according to
this system can be stored under refrigeration for over a year
without loss of performance due to acrylamide hydrolysis. Also,
stock buffers and stock gel solutions without polymerization
initiator can be stored for at least several weeks at room
temperature with no loss of performance.
[0085] In an embodiment of this gel formulation, an electrophoresis
gel is uniformly saturated with a gel buffer solution comprising a
primary organic amine or substituted amine with a pKa near
neutrality, and a submolar amount of acid or ampholyte buffer as
described above, so that the pH of the buffer is between about pH 6
and pH 8, preferably between about pH 6.5 to pH 7.5, and most
preferably 6.5 to 7.0. The electrophoresis gel may be any agarose
or polyacrylamide gel or a composite gel containing both agarose
and polyacrylamide. Preferably, the electrophoresis gel comprises
between 3% and 25% (% T) acrylamide polymerized using from about 1%
to about 6% cross linker (% C). More preferably, this
polyacrylamide gel is polymerized using from about 2% to about 5%
crosslinker (% C). Preferably, the amine comprises Bis-Tris or
N-(2-hydroxyethyl) morpholine, and most preferably, Bis-Tris.
Suitable acids and zwitterionic compounds are tricine, bicine,
N-(2-acetamido)-2-aminoethanesulfonic acid,
2-(N-morpholino)-2-hydroxypropanesulfonic acid,
N-tris-(hydroxymethyl)-2-ethanesulfonic acid,
N2-hydroxyethyl-piperazine-N2-ethanesulfonic acid,
N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid, and
3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic
acid.
[0086] In a gel and buffer system, current increases and migration
rates decline as the performance of the gels decline due to
hydrolysis of the polyacrylamide matrix. Any breakdown in a neutral
substance present at a large concentration, which produces a
charged species, will tend to disrupt the electrophoresis. This
disruption arises from the extra current produced, which in turn
increases joule eating without aiding the separation. In addition,
a discontinuity arises from the anionic and cationic hydrolysis
products forming in the gel that are not present in the cathode and
anode buffers. Hydrolysis of gel buffer species or additives takes
place independently from the gel matrix composition. The decrease
in migration rate may be attributed to higher fixed charge in the
gel caused by alkaline-catalyzed hydrolysis of the gel's
polyacrylamide. The fixed charge leads to significant counter-flow
of water, which can retard a macromolecule's migration rate. It has
been found that problems of gel instability producing lower
resolution, increased current, decreased migration rates can be
solved with gels buffered near neutrality and with buffer
substances having a pKa near neutrality. Such buffering systems
improve the performance of fresh or pre-cast polyacrylamide gels,
and fresh or pre-cast gels containing alkaline-labile materials,
such as urea or formamide, even when the gels are made with
base-stable polymers.
[0087] Without being bound by any one particular theory or
mechanism of action, it has been determined that it is advantageous
to use different buffer substances in the gel versus the anode and
cathode buffers. In some embodiments, the anionic substance used in
the gel or the cathode buffer need not be present in the anode
buffer since the anions do not migrate out of the anode buffer.
[0088] These buffer systems provide the benefits of a neutral pH
gel during storage and running, the least cost, and the fastest run
times.
[0089] A variety of methods for preparing the subject discontinuous
electrophoresis gels are known to those having ordinary skill in
the art, and any such methods may be employed to prepare an
electrophoresis gel without limitation. Described below is one
exemplary method that one skilled in the art may use to prepare a
discontinuous electrophoresis gel. It will be readily understood
however, that other methods of preparing discontinuous
electrophoresis gels may be equally employed, without departing
from the spirit and scope of the present embodiments.
[0090] In an embodiment, aqueous stock solutions of acrylamide and
bis-acrylamide may be prepared. Optionally, a single stock solution
of acrylamide and bis-acrylamide may be prepared. Additionally,
separate aqueous stock solutions of sucrose, a gel amine, a gel
ampholyte and a chloride containing acid may be prepared.
Optionally, separate stock solutions of SDS and reducing agent may
be prepared. Finally, stock solutions of ammonium persulfate (APS;
typically 10 wt. %) and tetramethylethylenediamine (TEMED;
typically between 4 wt. % to 10%) are prepared. Appropriate amounts
of the various stock solutions may be combined such that the final
composition has the desired concentration of each individual
component, except for the APS and TEMED, and the volume adjusted
with an appropriate diluent (e.g., water) such that the correct
concentration of each constituent is present. At this time, the APS
and TEMED are added so that polymerization of the acrylamide, and
bis-acrylamide is initiated. The mixture may then be cast between
to glass or plastic plates. The plates may be spaced from each
other such that the space between the plates in which the gel is
cast is between 2 mm to 0.5 mm apart. Additionally, the space
between the plates will be sealed on at least three sides.
Optionally, a thin layer of butanol or aqueous buffer may be
applied over the surface of the polymerizing gel, to reduce surface
tension of the polymerizing gel composition and ensure that the
upper edge of the polymerized gel is substantially straight. After
the polymerizing gel composition is cast between the plates, the
gel composition is allowed to rest for an appropriate amount of
time to achieve substantially complete polymerization of the
acrylamide and bis-acrylamide monomers.
[0091] In some embodiments, a stacking gel solution may be
separately prepared and cast over the polymerized resolving gel.
The S:R ratio of the fully polymerized electrophoresis gel will
typically be about 1:4 or about 1.9:7.5, though various other S:R
ratios may also be employed as described above and exemplified
below. After the stacking gel has been cast, but prior to the
polymerization reaction proceeding to completion, one or more combs
may be inserted into the aqueous polymerizing stacking gel
composition, between the two glass or plastic combs, thereby
defining a plurality of wells into which the sample may be added
immediately prior to use. Alternatively, in embodiments where no
stacking gel is used, the combs may be inserted into the
polymerizing aqueous resolving gel mixture described above.
Typically, though not necessarily, the combs may be kept in place
until the electrophoresis gel is ready for use, after which time
the comb is remove and the sample may be loaded into the well
defined by the polymerized polyacrylamide gel matrix.
[0092] In some embodiments, the percentage of polyacrylamide may
vary through the length of an electrophoresis gel. A variety of
such gradient gels are widely known in the art, any of which may be
adapted for use with the presently described formulations. Gradient
gels may be from about 2% to about 30% polyacrylamide, from about
3% to about 27% polyacrylamide, from about 4% to about 20%
polyacrylamide, or from about 5% to about 15% polyacrylamide. In
some embodiments, gradient gels may be from about 3% to about 12%
polyacrylamide, from about 3% to about 20% polyacrylamide, from
about 3% to about 15% polyacrylamide, from about 4% to about 12%
polyacrylamide, from about 4% to about 20% polyacrylamide, or from
about 4% to about 15% polyacrylamide. Any methods suitable for
preparing gradient polyacrylamide gels may be used to prepare
gradient gels having the subject formulations without limitation.
Such methods are widely known to one having ordinary skill in the
art to which the present embodiment pertains. Such methods are
disclosed in, e.g., U.S. Pat. Nos. 6,488,880; 6,267,579; 4,594,064;
and 5,071,531. In an embodiment, a gradient gel may optionally
include a stacking gel cast in accordance with the methods
discussed above and incorporated herein.
[0093] In some embodiments, the electrophoretic gel systems and
compositions may be packaged in air or water-tight packaging
following the polymerization of the gels. The air or water-tight
packaging may be specifically adapted to maximize moisture
retention by the cast gel and to prevent dehydration of the gel,
which may adversely affect the performance thereof. In an
embodiment, each gel may be individually packaged in air or
water-tight packaging and provided to a customer as part of a kit.
In an embodiment a kit may include a plurality of such individually
packaged electrophoretic gels. In an embodiment, a kit may include
2-100 individually packaged electrophoretic gels, 5-75 individually
packaged electrophoretic gels, 10-50 individually packaged
electrophoretic gels, 15-35 individually packaged electrophoretic
gels, or 20-25 individually packaged electrophoretic gels. The sets
of gels may themselves be packaged in a suitable container that is
shipped to an end user and is suitable to store the individually
packaged gels in an appropriate environment. Electrophoretic gels
prepared in accordance with the presently described compositions
and methods are storage stable for up to 6 month, up to 5 months,
up to 4 months, up to 3 months, up to 2 months or up to 1 month at
temperature ranges from about 1.degree. C. to about 25.degree. C.,
or from about 4.degree. C. to about 22.degree. C. In some
embodiments, a kit containing a plurality of individually packaged
electrophoretic separation gels may optionally be provided to a
customer in combination with one or more appropriate aqueous
running buffers. The aqueous running buffers may be provided at a
ready-to-use concentration, or may alternatively be provided in a
concentrated form that a user can dilute as need. The aqueous
running buffer may be provided, for example at a 20.times. stock
solution, a 15.times. stock solution, a 10.times. stock solution, a
5.times. stock solution or a 2.times. stock solution.
[0094] In an embodiment, an electrophoretic separation gel may be
assembled into an appropriate tank system that facilitates the
application of an electric field along the length of the gel. Such
a tank system will be configured to ensure that the aqueous running
buffer interacts with the gel and allows the voltage to be applied
equally along the entire longitudinal axis of the gel as disclosed
in detail above and incorporated herein.
[0095] The following examples are included to demonstrate some
preferred embodiments of the invention. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples which follow represent techniques discovered by the
inventor to function well in the practice of the invention, and
thus can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that one or more changes can be
made in the specific embodiments which are disclosed and still
obtain a like or similar result without departing from the spirit
and scope of the embodiments set forth herein.
EXAMPLES
[0096] The following experiments were performed to determine the
range of various constituents suitable for use in fast-running gel
formulations embodied herein. In particular, various combinations
of gel buffer constituents and salts were assessed. Unless
otherwise indicated, resolving polyacrylamide gels having the
compositions defined below were 8% polyacrylamide and 4.4%
bis-acrylamide cross-linker (8% T/4.4% C). Stacking gels having 4%
polyacrylamide and 3.8% bis-acrylamide cross-linker (4% T/3.8% C)
were hand-cast on top of the resolving gel. Unless otherwise
stated, the ratio of stacking gel to resolving gel (S:R) was about
1:4, the thickness of the gels was about 1 mm, and the gels were
hand-cast in mini gel cassettes.
[0097] The samples used to test various gel formulations described
below consisted of one or more of the following: Mark12.TM.,
SeeBlue Plus2.RTM. Pre-stained marker, Novex.RTM. Sharp
Pre-stained, and E. coli lysate (all obtained from Life
Technologies Corporation, Carlsbad, Calif.), in the indicated
lanes. Mark12.TM. was chosen due to its high salt concentration at
high loads (10 .mu.L) and its historical use as the standard of
choice for qualifying NuPAGE.RTM. gels. Novex.RTM. Sharp
Pre-stained standards were chosen to facilitate visual tracking of
the 3.5 kDa band corresponding to Insulin, chain B, so that
completion of the gel run can be easily monitored. SeeBlue
Plus2.RTM. was chosen because it is known that migration of the
19-22 kDa rhodamine-labeled myoglobin band can vary with amount of
voltage. For example, when SeeBlue Plus2.RTM. protein standards are
run on a NuPAGE.RTM. gel in turbo mode (i.e., 250 V), the myoglobin
band runs at a lower apparent molecular weight than if the gels
were run in normal mode (i.e., 200 V). E. coli lysate was chosen to
observe migration of a complex protein sample.
Example 1
[0098] The first test gel formulation examined was an 8% SDS-PAGE
resolving gel having the following composition: 100 mM Bis-Tris, 75
mM Tricine, 4 vol. % sucrose, and 20 mM BES. The stacking gel had
the following composition: 357 mM Bis-Tris and 210 mM Chloride, pH
6.5 cast on top of the resolving gel. The S:R ratio of the gel was
1:4.
[0099] Five (5) .mu.L of Mark12.TM. protein marker with Ponceau S
tracking dye was loaded in each lane of the gel and the gel was run
at 300 V for 15 minutes in NuPAGE.RTM. MES SDS Running Buffer
(Invitrogen Corp., Carlsbad, Calif.). Completion of the gel run was
determined based on the migration of Ponceau S tracking dye added
separately to each sample to the bottom of the gel. At the
completion of the run, the gel was stained with Coomassie.RTM.
G-250 using SimplyBlue.TM. SafeStain (from Invitrogen Corp,
Carlsbad, Calif.).
[0100] FIG. 1 shows the migration pattern of the marker bands
resulting from this gel formulation. The 3.5 kDa insulin B band
(indicated in FIG. 1) only migrated about 2/3 of the length of the
gel. This indicates that the (Ponceau S) tracking dye migrated
faster than the lower molecular weight proteins. Additionally, the
bands migrating faster than the 55.4 kDa glutamic dehydrogenase
band (indicated in FIG. 1) appear more diffuse than those migrating
slower than the 55.4 kDa band, and the outer edges of the 6 kDa
aprotinin band and the 3.5 kDa insulin B band are curved toward the
bottom of the gel. Thus, while the gel run time of 15 minutes is
improved, resolution of the bands using this gel formulation was
not optimal.
Example 2
[0101] An SDS-PAGE test gel was cast essentially as described in
Example 1, except that the S:R ratio was changed to 41:59 in an
attempt to improve the stacking and separation of bands migrating
higher than the 55.4 kDa glutamic dehydrogenase band. Five (5)
.mu.L of Mark12.TM. protein marker was loaded in each lane of the
gel and the gel was run at 300 V for 16 minutes in MES SDS Running
Buffer. Completion of the gel run was determined based on the
migration the Ponceau S tracking dye to the bottom of the gel and
the gel was stained with SimplyBlue.TM. SafeStain as above.
[0102] The resulting gel (FIG. 2) shows the migration pattern of
the marker bands resulting from this gel formulation. The 3.5 kDa
Insulin B band migrated the entire length of the gel, although an
additional 1 min of run time was required. The 200 kDa myosin band
in the outermost lanes (i.e., lanes 1, 2, 9 and 10; indicated by
the asterisk) was more diffuse than the corresponding band seen in
lanes 3-8. Additionally, bands migrating faster that the 55.4 kDa
glutamic dehydrogenase band (indicated in FIG. 2) were more diffuse
than those migrating slower than the 55.4 kDa band, and the outer
edges of the 6 kDa aprotinin band and the 3.5 kDa insulin B band
were curved toward the bottom of the gel.
Example 3
[0103] An SDS-PAGE test gel was cast essentially as described in
Example 2, with the following changes: the 8% (8% T/4.4% C)
resolving gel was made using 100 mM Bis-Tris and 100 mM Tricine.
The 4% (4% T/3.8% C) stacking gel cast over the resolving gel had a
pH of 6.1 and was made using 357 mM Bis-Tris and 150 mM sulfuric
acid. In this example, sulfuric acid was used in order to provide a
leading ion (SO.sub.4.sup.-2) that migrated slower than Cl.sup.-.
The S:R ratio of the gel was 41:59. Five (5) .mu.L of Mark12.TM.
protein marker was loaded in each lane of the gel and the gel was
run at 300 V for 18 minutes in MES SDS Running Buffer. Completion
of the gel run was determined based on the migration of the Ponceau
S tracking dye to the bottom of the gel after which the gel was
stained with SimplyBlue.TM. SafeStain as above.
[0104] FIG. 3 shows the migration pattern of the marker bands
resulting from this gel formulation. As shown, the use of sulfuric
acid in the stacking gel yielded unsatisfactory results and
appeared to overwhelm the buffering capability and resolution of
the MES buffer.
Example 4
[0105] Two identical 8% SDS-PAGE resolving gels were cast
essentially as described in Example 2, except that the amount of
Bis-Tris in each gel was reduced to 178 mM, the chloride was
reduced to 105 mM, and tricine was absent. A 4% stacking gel
comprising 178 mM Bis-Tris, 105 mM chloride having pH 6.5 was cast
on top of the each resolving gel. The S:R ratio of the gel was
1:3.
[0106] Five (5) .mu.l Mark12.TM. protein marker was loaded in lanes
1-3, 5, 7, 9 and 10 of each gel, 5 .mu.l SeeBlue Plus2.RTM.
Pre-Stained Protein Standard was loaded in lane 4, 10 .mu.g E. coli
lysate was loaded in lane 6, and 5 .mu.l Novex.RTM. Sharp
Pre-Stained Protein Standard was loaded in lane 8. One of the gels
(shown in FIG. 4A) was run in MES SDS Running Buffer at 300 V for
13 minutes, and the second gel (shown in FIG. 4B) was run in MES
running buffer at 350 V for 10 minutes. Completion of the gel run
was determined by visualizing migration of the pre-stained 3.5 kDa
band in lane 8. The gel was stained with SimplyBlue.TM. SafeStain
as above.
[0107] The 3.5 kDa insulin B band seen in FIGS. 4A and 4B shows
better migration characteristics than the gels depicted in FIGS.
1-3, although the bands are broader than the bands in lanes 4 and
8. Overall, for the gel shown in FIG. 4B that was run at 350 V, the
bands corresponding to the lower molecular weights were broader
than the corresponding bands in the gel shown in FIG. 4A that was
run at 300 V. Additionally, some band splitting is seen with the
bands in the 66-6 kDa range in the gel run at 350 V (FIG. 4B).
Example 5
[0108] An 8% SDS-PAGE resolving gel was cast essentially as
described in Example 1, except that the concentration of Bis-Tris
was increased to 228 mM, the concentration of chloride was adjusted
to 140 mM, the concentration of tricine was reduced to 50 mM, BES
was removed, and the pH of the resolving gel was 6.5. A 4% stacking
gel comprising 228 mM Bis-Tris, 140 mM Cl.sup.-, 50 mM Tricine, and
0 wt. % sucrose was cast on top of the resolving gel. Five (5)
.mu.l Mark12.TM. protein marker was loaded in lanes 1, 2, 5-7, 9
and 10, 5 .mu.l SeeBlue Plus2.RTM. Pre-Stained Protein Standard was
loaded in lane 3, 10 .mu.g E. coli lysate was loaded in lane 4, and
5 .mu.l Novex.RTM. Sharp Pre-Stained Protein Standard was loaded in
lane 8. The gel was run in MES SDS Running Buffer at 300 V for 13
minutes. Completion of the gel run was determined by visualizing
migration of the 3.5 kDa band in lane 8. The gel, shown in FIG. 5,
was stained with SimplyBlue.TM. SafeStain as above. Broadening of
the lower molecular weight bands was reduced, and band sharpness
was improved by the addition of 50 to 100 mM tricine and 120-160 mM
chloride.
Example 6
[0109] FIG. 6 demonstrates the differences in electrophoresis
characteristics between a commercially available SDS-PAGE gel
formulation (NuPAGE.RTM. Novex 8% Bis-Tris Midi gels; FIGS. 6A, 6C
and 6E) and the test gel formulation described in Example 5;
namely, an 8% SDS-PAGE resolving gel having 228 mM Bis-Tris, 140 mM
chloride and 50 mM tricine with 4% sucrose, and a 4% stacking gel
with the same buffer composition and no sucrose, where the S:R
ratio was 1.9:5.6 (indicated Test Gel in FIGS. 6B, 6D and 6F). The
following samples were prepared and loaded on each of the gels:
lanes 1, 2, 5, 6, 9 and 10 contained 5 .mu.l Mark12.TM. protein
marker, lane 7 contained 10 .mu.l Mark12.TM. protein marker, lane 3
contained 5 .mu.l SeeBlue Plus2.RTM. Pre-Stained Protein Standard,
lane 4 contained 10 .mu.g E. coli extract, and lane 8 contained 5
.mu.l Novex.RTM. Sharp Pre-Stained Protein Standard. The
NuPAGE.RTM. Novex 8% Bis-Tris gel and the test gel formulation were
run in MES SDS Running Buffer at 200 V for 32 minutes and 29
minutes, respectively (FIGS. 6A and 6B); at 250 V for 20 minutes
and 19 minutes, respectively (FIGS. 6C and 6D); and at 300 V for 16
minutes and 13 minutes, respectively (FIGS. 6E and 6F). After
completion of the gel run, the gels were stained with
SimplyBlue.TM. SafeStain as described above.
[0110] At 250V and 300V (FIGS. 6C-6F), the lower molecular weight
insulin B bands (indicated by arrows) showed sharper resolution and
shorter run times for the test gel formulation (FIGS. 6D and 6F)
than in NuPAGE.RTM. Novex 8% Bis-Tris (FIGS. 6C and 6E).
Additionally, at all voltages tested, the 14 and 17 kDa bands of
the SeeBlue Plus2.RTM. Pre-Stained Protein Standard showed better
resolution and separation in the test gel formulation (FIGS. 6B, 6D
and 6F, lane 3, boxed area) than in the NuPAGE.RTM. Novex 8%
Bis-Tris (FIGS. 6A, 6C and 6E, lane 3, boxed area).
Example 7
[0111] For the following experiments, test SDS-PAGE gels were cast
essentially as described in Examples 5 and 6, except as noted
below. For each set of experimental conditions, a total of 7 test
SDS-PAGE gels were cast. The test SDS-PAGE formulations were run at
300 V for the indicated time. In each experiment, two (2)
NuPAGE.RTM. Novex gels were tested for each of the indicated
conditions (200 V or 250V). All gels were run in MES SDS Running
Buffer for the indicated amount of time and at the indicated
voltage. At the end of the run, each gel was stained with
SimplyBlue.TM. SafeStain and imaged. The sharpness of each band was
determined using TOTALLAB.TM. software (Nonlinear Dynamics Ltd,
Durham, N.C.). Band sharpness was determined by measuring the area
of each band and dividing the area by the width of each well (well
width held constant 10 wells/gel). The migration of each band
(determined by its R.sub.f value) was determined using TOTALLAB.TM.
software and QC software.
[0112] In FIG. 7, 8% SDS-PAGE test gels as described in Example 6
were cast and compared with NuPAGE.RTM. Novex 8% Bis-Tris
formulation. FIG. 7A shows a representative image obtained of a
test SDS-PAGE gel run at 300 V for 14 minutes in MES buffer, where
lanes 1, 2, 4-7, 9 and 10 were loaded with 5 .mu.l Mark12.TM.
protein marker, lane 3 was loaded with 10 .mu.g E. coli lysate, and
lane 8 was loaded with 10 .mu.l Novex.RTM. Sharp Pre-Stained
Protein Standard. FIG. 7B shows a representative image of a 20 well
NuPAGE.RTM. Novex 8% Bis-Tris run at 200 V for 35 minutes in MES
buffer, where lanes 1, 3, 8, 10, 11, 13, 18 and 19 were loaded with
5 .mu.l Mark12.TM. protein marker, lane 5 was loaded with 10 .mu.g
E. coli lysate and lane 16 was loaded with 10 .mu.l Novex.RTM.
Sharp Pre-Stained Protein Standard. FIG. 7C shows a representative
image of an identical gel described in FIG. 7B, except that the gel
was run in MES SDS Running Buffer at 250 V for 22 minutes. FIG. 7D
is a bar graph summarizing the relative sharpness of each
individual band (measured by pixel area) of the Mark12.TM. protein
marker that was obtained for each experimental condition. Test gel
formulation run at 300V is shown as grey bars, NuPAGE.RTM. Novex 8%
Bis-Tris gel run at 200 V is shown as black bars, and NuPAGE.RTM.
Novex 8% Bis-Tris run at 250V using the Turbo protocol is shown as
white bars. The x-axis indicates which of the Mark12.TM. protein
marker bands is being analyzed, and the y-axis indicates pixel
area. FIG. 7E shows the average R.sub.f value obtained for each gel
formulation using either NonLinear Dynamics TOTALLAB.TM. v2003.02
software or QC Software as indicated.
[0113] The results shown in FIGS. 8A-8E were obtained essentially
as described for FIGS. 7A-7E with the following exceptions. The
polyacrylamide concentration in the test gels shown in FIG. 8A was
increased to 10% and the gels were run in MES buffer at 300 V for
16 minutes. In FIGS. 8B and 8C, 10-well NuPAGE.RTM. Novex 10%
Bis-Tris gels were used and the lanes were loaded as follows: lanes
1, 2, 5-6, 9 and 10 contained 5 .mu.l Mark12.TM. protein marker,
lane 3 contained 10 .mu.g E. coli lysate, and lane 8 contained 10
.mu.l Novex.RTM. Sharp Pre-Stained Protein Standard. In FIG. 8B,
the gel was run at 200 V in MES buffer for 36 minutes, and in FIG.
8C, the gel was run in MES buffer at 250 V for 24 minutes. The
analysis of the gels, shown in FIGS. 8D and 8E, was performed as
described for FIGS. 7D and 7E.
[0114] The results shown in FIG. 9A-9E were obtained essentially as
described for FIGS. 8A-8E with the following exceptions. The
SDS-PAGE test gel shown in 9A was cast as a 4-12% polyacrylamide
gradient gel run in MES buffer at 300V for 17 minutes. In FIGS. 9B
and 9C, 12-well NuPAGE.RTM. Novex 4-12% Bis-Tris gels were used and
the lanes were loaded as described for FIGS. 8B and 8C. In FIG. 9B,
the gel was run at 200 V in MES buffer for 37 minutes, and in FIG.
9C, the gel was run in MES buffer at 250 V for 25 minutes. The
analysis of the gels, shown in FIGS. 9D and 9E, was performed as
described for FIGS. 7D and 7E.
Example 8
[0115] For the following experiments shown in FIG. 10, two test
SDS-PAGE gels were cast essentially as described in Examples 6
through 8, except that the sucrose concentration in the resolving
gel shown in FIG. 10A was reduced to 2 wt. %, and the sucrose
concentration in the resolving gel shown in FIG. 10B remained at 4
wt. %. In FIG. 10A, the gel was run at 300 V for 15 minutes. In
FIG. 10B, the gel was run at 300 V for 26 minutes, FIG. 10C shows
the relative band sharpness obtained for the gels shown in FIGS.
10A and 10B.
[0116] In this patent, certain U.S. patents, U.S. patent
applications, and other materials (e.g., articles) have been
incorporated by reference. The text of such U.S. patents, U.S.
patent applications, and other materials is, however, only
incorporated by reference to the extent that no conflict exists
between such text and the other statements and drawings set forth
herein. In the event of such conflict, then any such conflicting
text in such incorporated by reference U.S. patents, U.S. patent
applications, and other materials is specifically not incorporated
by reference in this patent.
[0117] Further modifications and alternative embodiments of various
aspects of the invention may be apparent to those skilled in the
art in view of this description. Accordingly, this description is
to be construed as illustrative only and is for the purpose of
teaching those skilled in the art the general manner of carrying
out the invention. It is to be understood that the forms of the
invention shown and described herein are to be taken as the
presently preferred embodiments. Elements and materials may be
substituted for those illustrated and described herein, parts and
processes may be reversed, and certain features of the invention
may be utilized independently, all as would be apparent to one
skilled in the art after having the benefit of this description to
the invention. Changes may be made in the elements described herein
without departing from the spirit and scope of the invention as
described in the following claims. In addition, it is to be
understood that features described herein independently may, in
certain embodiments, be combined.
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