U.S. patent application number 15/005987 was filed with the patent office on 2016-07-28 for electrophoresis buffer for faster migration, improved resolution and extended shelf-life.
The applicant listed for this patent is DGel Electrosystem Inc.. Invention is credited to Pierre SEVIGNY.
Application Number | 20160216232 15/005987 |
Document ID | / |
Family ID | 44860708 |
Filed Date | 2016-07-28 |
United States Patent
Application |
20160216232 |
Kind Code |
A1 |
SEVIGNY; Pierre |
July 28, 2016 |
ELECTROPHORESIS BUFFER FOR FASTER MIGRATION, IMPROVED RESOLUTION
AND EXTENDED SHELF-LIFE
Abstract
There is provided an electrolyte solution for extending shelf
life, and/or accelerating or improving resolution or improving
transfer efficacy for blot applications, or accelerating and
improving resolution, or accelerating and improving transfer
efficacy of gel electrophoresis and containing
Tris(hydroxymethyl)aminomethane (TRIS), at least one zwitterion,
and water. The electrolyte solution may be used in buffer systems
for gel electrophoresis and the preparation of gels for gel
electrophoresis, such as Western blot.
Inventors: |
SEVIGNY; Pierre; (Montreal,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DGel Electrosystem Inc. |
Montreal |
|
CA |
|
|
Family ID: |
44860708 |
Appl. No.: |
15/005987 |
Filed: |
January 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14562648 |
Dec 5, 2014 |
9267916 |
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15005987 |
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13695005 |
Oct 26, 2012 |
9103779 |
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PCT/CA2011/000500 |
Apr 21, 2011 |
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14562648 |
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61440096 |
Feb 7, 2011 |
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61431757 |
Jan 11, 2011 |
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61373315 |
Aug 13, 2010 |
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61328208 |
Apr 27, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 27/44747 20130101;
G01N 27/44704 20130101; B01D 57/02 20130101 |
International
Class: |
G01N 27/447 20060101
G01N027/447 |
Claims
1.-43. (canceled)
44. An electrolyte solution comprising:
Tris(hydroxymethyl)aminomethane (TRIS); at least one zwitterion
chosen from 2-amino-2methyl-1,3-propanediol (AMPD),
N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid
(CAPSO),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
2-(cyclohexylamino)ethanesulfonic acid (CHES),
3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),
N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS),
4-(N-Morpholino)butanesulfonic acid (MOBS),
piperazie-N,N'-bis[2-hydroxypropanesulphonic]acid (POPSO),
N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),
(1,4-piperazinediethanesulphonic acid) (PIPES), and
Piperazine-N,N'-bis(3-propanesulfonic Acid) (PIPPS); water, and a
chelating agent having the name: ethylenediaminetetraacetate
(EDTA), ethylene glycol tetraacetic acid (EGTA), trisodium
nitrilotriacetate, hydroxyethyl ethylenediamine trisodium acetate
(trisodium HEDTA), diethylene triamino pentasodium acetate or
uramil disodium acetate, wherein said EDTA is at a concentration of
0.5% (w/v) or less; wherein the pH of the electrolyte solution is
from about 8.0 to about 8.8, wherein said electrolyte solution is
for at least one of: a shorter electrophoresis time of a gel
electrophoresis, an improved resolution of a gel electrophoresis,
an improved transfer efficacy of a gel electrophoresis, and a
shorter transfer time of a gel electrophoresis, compared to a
Tris-Glycine-SDS electrolyte solution at pH 8.3.
45. The electrolyte solution of claim 44, further comprising
methanol.
46. The electrolyte solution of claim 45, wherein said methanol is
up to 20%.
47. The electrolyte solution of claim 44, further comprising sodium
dodecyl sulphate (SDS).
48. The electrolyte solution of claim 47, wherein sodium dodecyl
sulphate (SDS) is 0.5% (wt/vol) or less.
49. The electrolyte solution of claim 48, wherein the concentration
of sodium dodecyl sulphate (SDS) is 0.1% (wt/vol) or less.
50. The electrolyte solution of claim 44, wherein a concentration
of said chelating agent is 0.5% (wt/vol) or less.
51. The electrolyte solution of claim 50, wherein a concentration
of said chelating agent is 0.05% (wt/vol) or less.
52. The electrolyte solution of claim 51, wherein a concentration
of said chelating agent is 0.03%.
53. The electrolyte solution of claim 44, wherein the concentration
of Tris(hydroxymethyl)aminomethane (TRIS) is from about 10 mM to
about 500 mM.
54. The electrolyte solution of claim 44, wherein the concentration
of the zwitterion is from about 1 mM to about 500 mM.
55. The electrolyte solution of claim 44, wherein the concentration
of Tris(hydroxymethyl)aminomethane (TRIS) is from about 50 mM to
about 150 mM.
56. The electrolyte solution of claim 44, wherein the concentration
of Tris(hydroxymethyl)aminomethane (TRIS) is from about 50 mM to
about 250 mM.
57. The electrolyte solution of claim 44, wherein the concentration
of Tris(hydroxymethyl)aminomethane (TRIS) is 150 mM.
58. The electrolyte solution according to claim 44, wherein said
zwitterions is a combination of
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES), and
(1,4-piperazinediethanesulphonic acid) (PIPES).
59. An electrophoresis gel comprising the electrolyte solution of
claim 44, wherein the gel is chosen from an acrylamide gel at a
concentration from less than 1% (wt/vol) to about 25% (wt/vol), an
agarose gel at a concentration from about 0.5% (wt/vol) to about 3%
(wt/vol) and a combination thereof.
60. The electrophoresis gel of claim 59, further comprising sodium
dodecyl sulphate (SDS).
61. The electrophoresis gel of claim 59, wherein the pH is from
about 8.0 to about 8.8.
62. A method of accelerating or improving the resolution, or
improving transfer efficacy, or accelerating and improving the
resolution, or accelerating and improving transfer efficacy of the
electrophoretic separation of at least one sample comprising the
step of: applying a voltage to an electrolyte solution according to
claim 44, in contact with an electrophoresis gel containing the at
least one sample therein.
Description
BACKGROUND
[0001] (a) Field
[0002] The subject matter disclosed generally relates to gel
electrophoresis. More specifically, the subject matter disclosed
relates to an electrolyte solution for accelerating or improving
resolution, or accelerating and improving resolution of gel
electrophoresis and containing Tris(hydroxymethyl)aminomethane
(TRIS), at least one zwitterion, and water.
[0003] (b) Related Prior 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, sandwiched as a slab between glass or
plastic plates, or poured in a plastic tray. The gel has an open
molecular network structure, defining pores which are saturated
with an electrically conductive buffered solution. 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.
[0007] The most common buffer system employed for the separation of
proteins is the Laemmli buffer system consists of 0.375 M tris
(hydroxy methyl) 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. Many different gel
separation materials have been disclosed, with different
compositions, pH characteristics, voltage requirements, etc. The
goal of most of the recent innovations in the field has been to
provide an electrophoresis gel which can be used to perform a
faster, more accurate, more stable, or therefore more versatile
electrophoresis.
[0008] A number of different gel buffer systems have been proposed
for use at or around neutral pH that do not involve the use of the
Tris-HCl Glycine buffer system of Laemmli.
[0009] For example, U.S. Pat. No. 6,096,182 to Updyke et al.
discloses an electrophoresis gel at a neutral pH. The advantage of
producing such a gel is that the gel system is stable, with reduced
reactivity and increased shelf life.
[0010] U.S. Pat. No. 5,464,517 to Hjerten et al. discloses an
electrophoresis buffer which has a high buffering capacity and low
electrical conductivity. The advantage of this type of buffer,
particularly in capillary electrophoresis, is that it allows the
separation to be performed at a higher voltage and consequently
more quickly.
[0011] A majority of innovations have focused on improving
electrophoresis by proposing new recipes for the gel buffer.
[0012] Therefore, there is a need for reagents that will improve
the speed at which electrophoresis can be performed, improve gel
resolution, as well as increase the shelf life of gels.
SUMMARY
[0013] In a first embodiment there is disclosed an electrolyte
solution for accelerating, or improving resolution, or improving
transfer efficacy for blot applications, including Western blot for
example, or accelerating and improving resolution, or accelerating
and improving Western transfer efficacy of gel electrophoresis
containing: [0014] Tris(hydroxymethyl)aminomethane (TRIS); [0015]
at least one zwitterion that may be chosen from
2-amino-2methyl-1,3-propanediol (AMPD),
N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid
(CAPSO),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
2-(cyclohexylamino)ethanesulfonic acid (CHES),
3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),
N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS),
4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholino
propanesulfonic acid (POPSO),
N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES)), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),
(1,4-piperazinediethanesulphonic acid) (PIPES),
(3-[N-morpholino]propanesulphonic acid)(MOPS), and
Piperazine-N,N'-bis(3-propanesulfonic Acid) (PIPPS); and [0016]
water.
[0017] The TRIS component of the above electrolyte solution may be
selected from the group consisting of: Tris-HCl Glycine,
Tris-Glycine, MOPS-Bis-Tris, EPPS-Tris-SDS, and the like.
[0018] The zwitterion may be chosen from
N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
2-(cyclohexylamino)ethanesulfonic acid (CHES), N-Glycylglycine
(Gly-Gly), 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid
(EPPS or HEPPS),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES)), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),
(1,4-piperazinediethanesulphonic acid) (PIPES),
(3-[N-morpholino]propanesulphonic acid)(MOPS), and
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) for
accelerating gel electrophoresis.
[0019] The zwitterion may be chosen from
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid
(CAPSO), N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),
(1,4-piperazinediethanesulphonic acid) (PIPES),
(3-[N-morpholino]propanesulphonic acid)(MOPS), and
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) for
improving resolution of gel electrophoresis.
[0020] The zwitterion may be chosen from N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS),), N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid
(BES),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),
(1,4-piperazinediethanesulphonic acid) (PIPES),
(3-[N-morpholino]propanesulphonic acid)(MOPS), and
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) for
accelerating and improving resolution of gel electrophoresis.
[0021] The zwitterion may be chosen from N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO)
for accelerating and improving Western transfer efficacy of gel
electrophoresis.
[0022] The zwitterions may be chosen from N-Glycylglycine
(Gly-Gly), 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid
(EPPS or HEPPS), N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic
acid (BES), and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic
acid (CAPSO) can also be used in combination with one or more other
zwitterions from the group 2-amino-2methyl-1,3-propanediol (AMPD),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
2-(cyclohexylamino)ethanesulfonic acid (CHES),
3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),
N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS),
4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholino
propanesulfonic acid (POPSO),
N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES) and Piperazine-N,N'-bis(3-propanesulfonic Acid) (PIPPS)
and keep its accelerating efficacy in electrophoresis separation
and Western transfer as well as providing improved separation
resolution.
[0023] The zwitterions for improving resolution may be
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS).
[0024] The electrolyte solution may have a pH from about 8.0 to
about 8.8.
[0025] The electrolyte solution may comprise methanol, up to
20%.
[0026] The electrolyte solution may comprise sodium dodecyl
sulphate (SDS) or lithium dodecyl sulfate (LDS), up to 5%.
[0027] The electrolyte solution may comprise a chelating agent
having the name: ethylenediaminetetraacetate (EDTA), ethylene
glycol tetraacetic acid (EGTA), trisodium nitrilotriacetate,
hydroxyethyl ethylenediamine trisodium acetate (trisodium HEDTA),
diethylene triamino pentasodium acetate or uramil disodium
acetate.
[0028] The concentration of Tris(hydroxymethyl)aminomethane (TRIS)
may be from about 10 mM to about 500 mM, from about 50 mM to about
150 mM, or from about 50 mM to about 250 mM, or may be 150 mM.
[0029] The concentration of the zwitterion may be from about 1 mM
to 500 mM, from about 10 mM to about 500 mM, or from about 25 mM to
about 50 mM, or from about 50 mM to about 100 mM, or may be 50 mM
or may be 100 mM.
[0030] The concentration of sodium dodecyl sulphate (SDS) may be
0.5% (wt/vol) or less, or may be 0.1% (wt/vol) or less, or may be
0.1% (wt/vol).
[0031] The concentration of ethylenediaminetetraacetate (EDTA) may
be 0.5% (wt/vol) or less, or may be 0.05% (wt/vol) or less or may
be 0.03% (wt/vol).
[0032] The zwitterions may be a combination of
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),
(1,4-piperazinediethanesulphonic acid) (PIPES), and
(3-[N-morpholino]propanesulphonic acid)(MOPS).
[0033] In a second embodiment, there is disclosed an
electrophoresis gel comprising the electrolyte solution according
to the present invention. The gel may be an acrylamide gel, and the
concentration of acrylamide may be from about 4% (wt/vol) to about
25% (wt/vol). The gel may be an agarose gel, and the concentration
of agarose may be from about 0.5% (wt/vol) to about 3% (wt/vol).
The gel may be an acrylamide and agarose gel, and the concentration
of acrylamide may be from about less than 1% (wt/vol) to about 25%
(wt/vol) and the concentration of agarose may be from about 0.5%
(wt/vol) to about 3% (wt/vol). The electrophoresis gel may comprise
sodium dodecyl sulphate (SDS).
[0034] The zwitterion may be chosen from
2-amino-2methyl-1,3-propanediol (AMPD),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),
2-(cyclohexylamino)ethanesulfonic acid (CHES),
N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic
acid (TAPS) and 3-N-Morpholino propanesulfonic acid (POPSO) for
improving shelf life of the gel.
[0035] The pH of the gel may be from about 8.0 to about 8.8.
[0036] In yet another embodiment, there is disclosed a method of
accelerating or improving the resolution, or accelerating and
improving the resolution of the electrophoretic separation of at
least one sample comprising the step of applying a voltage to an
electrolyte solution according to the present invention, in contact
with an electrophoresis gel containing the at least one sample
therein.
[0037] The electrophoresis gel is a gel according to the present
invention.
[0038] In yet another embodiment, there is disclosed a method of
accelerating or improving the resolution of, or improving transfer
efficacy, or accelerating and improving the resolution, or
accelerating and improving transfer efficacy of the electrophoretic
separation of at least one sample comprising the step of applying a
voltage to an electrolyte solution adapted to perform gel
electrophoresis in contact with an electrophoresis gel according to
the present invention containing the at least one sample
therein.
[0039] In yet another embodiment, there is disclosed a method for
improving resolution of an electrophoretic separation of at least
one sample comprising adding at least one zwitterion chosen from
2-amino-2methyl-1,3-propanediol (AMPD),
N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid
(CAPSO),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
2-(cyclohexylamino)ethanesulfonic acid (CHES),
3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),
N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS),
4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholino
propanesulfonic acid (POPSO),
N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),
(1,4-piperazinediethanesulphonic acid) (PIPES),
(3-[N-morpholino]propanesulphonic acid)(MOPS), and
Piperazine-N,N'-bis(3-propanesulfonic Acid) (PIPPS) to an
electrophoresis buffer or to an electrophoresis gel, or to both an
electrophoresis buffer and an electrophoresis gel. The zwitterion
may be 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS).
[0040] In yet another embodiment, there is disclosed a use of an
electrolyte solution according to the present invention for
accelerating gel electrophoresis.
[0041] In yet another embodiment, there is disclosed a use of an
electrolyte solution according to the present invention for
Improving resolution of gel electrophoresis.
[0042] In yet another embodiment, there is disclosed a use of an
electrolyte solution according to the present invention for
accelerating and improving resolution of gel electrophoresis.
[0043] In yet another embodiment, there is disclosed a use of a gel
according to the present invention for accelerating or improving
resolution, or accelerating and improving resolution of gel
electrophoresis.
[0044] In yet another embodiment, there is disclosed a use of an
electrolyte solution according to the present invention for the
preparation of an electrophoresis gel, wherein the zwitterions is a
compound of formula: 2-amino-2methyl-1,3-propanediol (AMPD),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),
2-(cyclohexylamino)ethanesulfonic acid (CHES),
N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic
acid (TAPS) or 3-N-Morpholino propanesulfonic acid (POPSO) for
improving shelf life of a gel.
[0045] The following terms are defined below.
[0046] The term "improved resolution" is intended to mean a better
resolution which allows separation of sharper or narrower bands of
molecules, distanced or spaced apart from each other as opposed to
other means of separation which have broader or thicker bands. This
facilitates physical separation or molecular weight identification
of the different molecules that make up these bands over the entire
range of molecular weight.
[0047] Features and advantages of the subject matter hereof will
become more apparent in light of the following detailed description
of selected embodiments, as illustrated in the accompanying
figures. As will be realized, the subject matter disclosed and
claimed is capable of modifications in various respects, all
without departing from the scope of the claims. Accordingly, the
drawings and the description are to be regarded as illustrative in
nature, and not as restrictive and the full scope of the subject
matter is set forth in the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 illustrates polyacrylamide gels electrophoresed in
electrolyte solutions according embodiments of the present
invention and compared to a gel electrophoresed on Tris-glycine-SDS
(baseline) solution.
[0049] FIG. 2 illustrates polyacrylamide gels electrophoresed in
electrolyte solution according an embodiment of the present
invention.
[0050] FIG. 3 illustrates polyacrylamide gels prepared with
electrolyte solutions according to embodiments of the present
invention, and kept at 4.degree. C. to age during 360 days. Each
month, gels were run to measure changes in resolution, migration
path and migration speed.
[0051] FIG. 4 illustrates membranes comprising prestained protein
marker that have been transferred from polyacrylamide gels by (A)
wet transfer, (B) semi-dry transfer, (C) asymmetric buffer for a
"dry" transfer, and (D) a control semi-dry transfer using the
classic Towbin buffer, run 275 mA for 15 minutes. The transfer in D
is not as efficient. The run prestained protein markers range from
6 KDa to 245 KDa.
[0052] FIG. 5 illustrates a prestained protein marker that have
been transferred from polyacrylamide gels in the eStain.TM.
apparatus using a 5.times. solution of an EPPS (or HEPPS)
containing buffer according to the present invention.
[0053] FIG. 6 (A-L) illustrates a comparison of the migration
resolution of MOPS based gels in an electrolyte solutions according
to the present invention vs. the regular running buffer for the
MOPS based gels.
[0054] FIG. 7 (A-C) illustrates a comparison of the migration
resolution of 10% acrylamide Tris-glycine based gel (A), 4-20%
acrylamide Tris-glycine based gel (B), and 10% acrylamide EPPS
based gel, in an electrolyte solutions according to the present
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0055] The present inventors have now surprisingly found that
selecting specific zwitterions to prepare an electrolyte solution
for use in gel electrophoresis can result in unexpected increase in
electrophoresis speed, improvement in gel resolution, or both.
These improvements may be observed when the electrolyte solutions
are used as running buffer (also referred to as "reservoir" buffer)
for the electrophoresis apparatus, as well as when used in as
buffer system for the preparation of the electrophoresis gels (i.e.
in the gel). Furthermore, when used in the preparation of
electrophoresis gels, some of these zwitterions will also increase
in the shelf life of gels prepared using these electrolyte
solutions.
[0056] In embodiments there are disclosed an electrolyte solution
for performing gel electrophoresis. The electrolyte contains
specific components.
[0057] Zwitterions
[0058] A zwitterion is a chemical compound that carries a total net
charge of 0 and is thus electrically neutral, but carries formal
charges on different atoms. Zwitterions are polar and are usually
very water-soluble, but poorly soluble in most organic solvents.
Zwitterions will exist mostly as zwitterions in a certain range of
pH. The pH at which the average charge is zero is known as the
molecule's isoelectric point.
[0059] The zwitterions of interest in the present invention belong
to the category commonly referred to as biological buffers, which
are buffers that are commonly used as buffering agents in
biological laboratories. Examples of biological buffers that can be
cited are those known as bis-TRIS
(2-bis[2-hydroxyethyl]amino-2-hydroxymethyl-1,3-propanediol), ADA
(N-[2-acetamido]-2-iminodiacetic acid), ACES
(2-[2-acetamino[-2-aminoethanesulphonic acid). PIPES
(1,4-piperazinediethanesulphonic acid), MOPSO
(3-[N-morpholino]-2-hydroxypropanesulphonic acid), bis-TRIS PROPANE
(1,3-bis[tris(hydroxymethyl)methylaminopropane]), BES
(N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid), MOPS
(3-[N-morpholino]propanesulphonic acid), TES
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid), HEPES
(N-[2-hydroxyethyl]piperazine-N'-(2-ethanesulphonic)acid), DIPSO
(3-N,N-bis[2-hydroxyethyl]amino-2-hydroxypropanesulphonic acid),
MOBS (4-N-morpholinobutanesulphonic acid), TAPSO
(3[N-tris-hydroxymethyl-methylamino]-2-hydroxypropanesulphonic
acid), TRIS (2-amino-2-[hydroxymethyl]-1,3-propanediol), HEPPSO
(N-[2-hydroxyethyl]piperazine-N'-[2-hydroxypropanesulphonic]acid),
POPSO (piperazine-N,N'-bis[2-hydroxypropanesulphonic]acid), TEA
(triethanolamine), EPPS (or HEPPS)
(N-[2-hydroxyethyl]-piperazine-N'-[3-propanesulphonic]acid),
TRICINE (N-tris[hydroxymethyl]methylglycine), GLY-GLY (diglycine),
BICINE (N,N-bis[2-hydroxyethyl]glycine), HEPBS
(N-[2-hydroxyethyl]piperazine-N'-[4-butanesulphonic]acid), TAPS
(N-tris[hydroxymethyl]methyl-3-aminopropanesulphonic acid), AMPD
(2-amino-2-methyl-1,3-propanediol), TABS
(N-tris[hydroxymethyl]methyl-4-aminobutanesulphonic acid), AMPSO
(3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulphonic
acid), CHES (2-(N-cyclohexylamino)ethanesulphonic acid), CAPSO
(3-[cyclohexylamino]-2-hydroxy-1-propanesulphonic acid), AMP
(2-amino-2-methyl-1-propanol), CAPS
(3-cyclohexylamino-1-propanesulphonic acid), and CABS
(4-[cyclohexylamino]-1-butanesulphonic acid).
[0060] Although the biological buffering property of these
zwitterions has been recognized, the capacity of a select group of
these zwitterions to positively impact the performance of gel
electrophoresis, and the shelf life of gels for gel electrophoresis
was not.
[0061] Preferably the zwitterions include ACES
(2-[2-acetamino[-2-aminoethanesulphonic acid), PIPES
(1,4-piperazinediethanesulphonic acid), MOPS
(3-[N-morpholino]propanesulphonic acid),
2-amino-2methyl-1,3-propanediol (AMPD), N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid
(CAPSO),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
2-(cyclohexylamino)ethanesulfonic acid (CHES), N,
N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES), 3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic
acid (DIPSO), 4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid
(EPPS or HEPPS). N-Glycylglycine (Gly-Gly),
N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS),
4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholino
propanesulfonic acid (POPSO),
N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) and
Piperazine-N,N'-bis(3-propanesulfonic Acid) (PIPPS). These
zwitterions significantly improve the speed at which
electrophoresis may be performed, improve gel resolution, or both.
The zwitterions may improve resolution by improving the definition
(or sharpness), by providing thinner or narrow bands) of the
molecules separated, by improving the separation between the
molecules (i.e. the distance between the individual bands, or by
both improving the definition (or sharpness) and separation of the
molecules. Also, when used for the preparation of gels, increase in
the shelf life of gels prepared using these electrolyte
solutions
[0062] The ranges of concentration over which these zwitterions may
be used for the preparation of electrolyte solutions according to
the present invention, for accelerating gel electrophoresis and/or
improving gel resolution discussed herein are from about 1 mM to
about 500 mM, or from about 10 mM to about 500 mM, or from about 25
mM to about 50 mM, or from about 25 mM to about 100 mM, or from
about 10 mM to about 100 mM, or from about 1 mM to about 100 mM, or
from about 1 mM to about 75 mM, or from about 10 mM to about 75 mM,
or from about 1 mM to about 10 mM, or from about 1 mM to about 50
mM or from about 10 mM to about 50 mM.
[0063] The ranges of concentration over which these zwitterions may
be used for improving the shelf life of gels prepared according to
the present invention are from about 1 mM to about 500 mM, or from
about 10 mM to about 500 mM, or from about 10 mM to about 100 mM,
or from about 1 mM to about 100 mM, or from about 1 mM to about 75
mM, or from about 10 mM to about 75 mM, or from about 1 mM to about
10 mM, or from about 1 mM to about 50 mM or from about or from
about 10 mM to about 50 mM or, from about 25 mM to about 375 mM,
and preferably, at 100 mM.
[0064] Preferably, the zwitterions to accelerate gel
electrophoresis (as compared to the classical Tris-Glycine-SDS at
pH 8.3 of Laemmli) are 2-amino-2methyl-1,3-propanediol (AMPD),
N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
2-(cyclohexylamino)ethanesulfonic acid (CHES), N-Glycylglycine
(Gly-Gly), 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid
(EPPS or HEPPS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic
acid (TAPS), ACES (2-[2-acetamino[-2-aminoethanesulphonic acid),
PIPES (1,4-piperazinediethanesulphonic acid), MOPS
(3-[N-morpholino]propanesulphonic acid), and
(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic
acid) (TES). When using the above zwitterions in the electrolyte
solutions according to the present invention, for running and/or
preparing an electrophoresis gel, the migration speed of gels may
be increase by about 30% or more. The zwitterions may be used alone
or in combination.
[0065] Optimal results with
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS)
are obtained at pH of about pH 8.0 to 8.8.
[0066] Preferably the zwitterions to improve the resolution of gels
during electrophoresis (as compared to the classical
Tris-Glycine-SDS at pH 8.3 of Laemmli) are
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid
(BES), N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), 3-N-Morpholino propanesulfonic acid (POPSO) ACES
(2-[2-acetamino[-2-aminoethanesulphonic acid), PIPES
(1,4-piperazinediethanesulphonic acid), MOPS
(3-[N-morpholino]propanesulphonic acid), and
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS).
When using the above zwitterions in the electrolyte solutions
according to the present invention, for running and/or preparing an
electrophoresis gel, the resolution of the gels may be increase by
40% or more. Most preferably the zwitterion to improve the
resolution of gels during electrophoresis is
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS). The resolution may be improved by improving the definition
(or sharpness), by providing thinner or narrow bands) of the
molecules separated, by improving the separation between the
molecules (i.e. the distance between the individual bands, or by
both improving the definition (or sharpness) and separation of the
molecules. According to one embodiment of the present invention,
the improvement in resolution may result in a normal, continuous
acrylamide concentration gel showing characteristic similar to a
gradient concentration of acrylamide gel. These gradient-like
improvements in resolution offer better and greater separation
between bands of molecules, while maintaining or improving
sharpness of the protein bands.
[0067] Preferably, the zwitterions to accelerate gel
electrophoresis and to improve the resolution of gels during
electrophoresis (as compared to the classical Tris-Glycine-SDS at
pH 8.3 of Laemmli) are
N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),
N-Glycylglycine (Gly-Gly),
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),
ACES (2-[2-acetamino[-2-aminoethanesulphonic acid), PIPES
(1,4-piperazinediethanesulphonic acid), MOPS
(3-[N-morpholino]propanesulphonic acid), and
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS). When using the above zwitterions in the electrolyte
solutions according to the present invention, for running and/or
preparing an electrophoresis gel, the resolution of the gels may be
increase by 40% or more and the migration speed of gels may be
increase by about 30% or more. Most preferably the zwitterion to
accelerate gel electrophoresis and to improve the resolution of
gels during electrophoresis is
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS). The resolution may be improved by improving the definition
(or sharpness), by providing thinner or narrow bands) of the
molecules separated, by improving the separation between the
molecules (i.e. the distance between the individual bands, or by
both improving the definition (or sharpness) and separation of the
molecules.
[0068] Preferably the zwitterions to improve the shelf life of gels
(as compared to the classical Tris-Glycine-SDS gels of Laemmli) are
2-amino-2methyl-1,3-propanediol (AMPD),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), N-Glycylglycine (Gly-Gly),
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or
HEPPS), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
2-(cyclohexylamino)ethanesulfonic acid (CHES), and 3-N-Morpholino
propanesulfonic acid (POPSO).
[0069] Optimal results with
4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS)
are obtained at pH of about pH 8.0 to 8.8.
[0070] Tris(Hydroxymethyl)Aminomethane Base (TRIS)
[0071] Tris is an abbreviation of the organic compound known as
tris(hydroxymethyl)aminomethane, with the formula
(HOCH.sub.2).sub.3CNH.sub.2. Tris is extensively used in
biochemistry and molecular biology. In biochemistry, Tris is widely
used as a component of buffer solutions, such as in TAE and TBE
buffer, especially for solutions of nucleic acids.
[0072] Tris also allows the pH of the electrolyte solution of the
present invention to be set towards more basic pH values. Tris also
has a positive impact on the migration speed in the preferred
concentration ranges.
[0073] The ranges of concentration over which Tris base may be used
for the preparation of electrolyte solutions according to the
present invention, for accelerating gel electrophoresis and
improving gel resolution as discussed herein are from about 10 mM
to about 500 mM. The Tris concentration has a direct impact on the
pH of the solution. An increasing amount of Tris increases the pH.
This allow fine adjustments in adapting the electrolyte solution of
the present invention to different gel chemistry and keeping a
proper pH ratio compatible with gel and buffer. Preferably the
concentration of Tris is from about 50 mM to about 150 mM, or from
about 50 mM to about 250 mM and most preferably, at about 150
mM.
[0074] The ranges of concentration over which Tris base may be used
for improving the shelf life of gels prepared according to the
present invention is from about 50 mM to about 375 mM, and
preferably 150 mM.
[0075] Sodium Dodecyl Sulfate or Other Anionic Surfactants
[0076] Sodium dodecyl sulfate (SDS) (C.sub.12H.sub.25SO.sub.4Na) is
an anionic surfactant is commonly used in preparing proteins for
electrophoresis in the SDS-PAGE technique. The molecule has a tail
of 12 carbon atoms, attached to a sulfate group, giving the
molecule the amphiphilic properties required of a detergent.
[0077] SDS may be optionally added to the electrolyte solution of
the present invention. The superior results obtained with the
electrolyte solutions according to the present invention are
obtained independently of the presence of SDS. The ranges of
concentration over which SDS may be used for the preparation of
electrolyte solutions according to the present invention, for all
the applications discussed herein are from 0.5% or less.
Preferably, from about 0.1% or less, and most preferably, at
0.1%.
[0078] Other anionic surfactants may be included in the electrolyte
solution of the present invention, such as, in a non-limiting
manner facultative, one anionic surfactant to provide to the buffer
denaturing properties for protein analysis: SDS, sodium dodecyl
sulphate, sodium lauryl sulfate (SLS), sodium laurilsulfate or
sodium, NaDS. The ranges of concentration over which they may be
used for the preparation of electrolyte solutions according to the
present invention, for all the applications discussed herein are
from 1.0% or less. Preferably, from about 0.1% or less, and most
preferably, at about 0.1%.
[0079] Chelating Agents
[0080] Ethylenediaminetetraacetate (EDTA) has a role as a chelating
agent, i.e. its ability to "sequester" metal ions such as Ca.sup.2+
and Fe.sup.3+. After being bound by EDTA, metal ions remain in
solution but exhibit diminished reactivity. Other chelating agents
may include ethylene glycol tetraacetic acid (EGTA), trisodium
nitrilotriacetate, hydroxyethyl ethylenediamine trisodium acetate
(trisodium HEDTA), diethylene triamino pentasodium acetate or
uramil disodium acetate.
[0081] EDTA may be optionally added to the electrolyte solution of
the present invention. The ranges of concentration over which EDTA
may be used for the preparation of electrolyte solutions according
to the present invention, for all the applications discussed herein
are from 0.5% or less. Preferably, from about 0.05% or less, and
most preferably, at 0.03%.
[0082] Methanol
[0083] In electrophoresis for transfer onto membranes, methanol is
normally added to the transfer buffer to slow the protein migration
and normally permit a more equal protein transfer between the high
molecular weight proteins and the low molecular weight. In standard
protocol, it is used as 10% or 20% of methanol. The efficacy of
fast transfer with electrolyte solutions according to the present
invention containing 0% to 20% methanol is working equally well to
100% efficacy in transfer under all these conditions.
[0084] Use of the Electrolyte Solutions
[0085] In use the electrolyte solutions of the present invention is
compatible with a very wide range of other buffer systems. The
electrolyte solution of the present invention may be used to run
electrophoresis of gels of any type, prepared with the same or with
different buffer systems than that of the present invention, even
including gels using different chemistries, such as MOPS as a
buffer (such as those described in US Patent publication No.
20060118418), under the appropriate conditions.
[0086] According to one embodiment, electrolyte solution according
to the present invention containing Tris and the zwitterions EPPS
(e.g. from about 10 mM to about 100 mM), ACES (e.g. from about 10
mM to about 75 mM), MOPS (e.g. from about 10 mM to about 50 mM) and
PIPES (e.g. from about 1 mM to about 10 mM) may be used to improve
resolution and migration speed of gels based on different
chemistries than Leammli's. An anionic surfactant (such as SDS from
about 0% to about 1%) may be included in such buffers to provide
denaturing properties. The resolution may be improved by improving
the definition (or sharpness), by providing thinner or narrow
bands) of the molecules separated, by improving the separation
between the molecules (i.e. the distance between the individual
bands, or by both improving the definition (or sharpness) and
separation of the molecules.
[0087] According to the present invention, electrophoresis includes
the separation of samples of DNA or protein or any other type of
molecule that may be separated accordingly, as well as their
transfer onto membranes or other suitable solid support such has
nitrocellulose, nylon, PVDF or other types of membranes that are
commonly used for applications such as Western transfer and
blotting.
[0088] The electrolyte solution according to the present invention
may be used in transfer systems of commercial make (e.g. wet,
semi-dry or dry transfer systems). For examples, the electrolyte
solution according to the present invention may be employed in
semi-dry or dry transfer systems such as Invitrogen XCell II.TM.
Blot Module, Pierce.TM. Fast Western System, Bio-Rad's Trans-Blot
SD.TM. Semi-Dry Electrophoretic Transfer Cell and equivalent
equipment. Liquid (i.e. wet) systems such as Bio-Rad Mini
Trans-Blot.RTM. Module, GE MiniVe Blot module and other
equivalents. Electrophoresis systems such as the eSTain.TM. Protein
Staining System from GenScript, which comprise both the
electrophoresis unit and the power supply may also work with the
electrolyte solution according to the present invention. Other
equivalent systems also functions as well.
[0089] The electrolyte solution of the present invention may be
used as the buffer system in most of gels used in molecular biology
and biochemistry, as described in classical references such as:
Uriel 1966, Bull. Soc. Chem. Biol. 48:969; Peacock & Dingman
1967, Biochem 6(6), 1818-1827; Peacock & Dingman 1968, Biochem
7(2), 668-674; Gaal, Electrophoresis in the separation of
biological macromolecules, p 422, Wiley, 1980. The electrolyte
solutions of the present invention may be included in acrylamide
gels (polyacrylamide gels), under native (without SDS) or
denaturing conditions (with SDS) that are typically prepared with
acrylamide concentrations from about 4% to about 25%. The
electrolyte solutions of the present invention may be included in
agarose gels that are typically prepared with agarose
concentrations from about 0.5% to about 3%.
[0090] EPPS (or HEPPS), TAPS, TES, BES or Glygly can be added to
the Laemmli buffer Tris-Glycine-SDS at a working concentration of
25 mM to 150 mM and preferably at 50 mM and accelerate the
migration efficacy of the Laemmli buffer by about 30%. In
electrophoresis for transfer onto membranes, methanol is normally
added to the transfer buffer to slow the protein migration and
normally permit a more equal protein transfer between the high
molecular weight proteins and the low molecular weight. In standard
protocol, it is used as 10% or 20% of methanol. The efficacy of
fast transfer with electrolyte solutions according to the present
invention containing 0% to 20% methanol is working equally well to
100% efficacy in transfer under all these conditions.
ALTERNATIVE EMBODIMENTS
Example I
[0091] Exemplary electrolyte solutions compositions were prepared
to be used with polyacrylamide or agarose gels made using classic
recipes such as the Laemmli buffer system (Tris-Glycine-SDS). The
exemplary electrolyte solutions were prepared and tested using a
common basic recipe:
[0092] Zwitterions, 50 mM, Tris (Base), 100 mM, SDS 0.1%, and EDTA
0.03%. For each prepared solution, the pH is measured, and if the
value was outside of the pH 7.9 to 8.6 range, it was adjusted with
HCl to acidify, or NaOH to make more basic.
[0093] 500 ml of running buffer were prepared for each zwitterions,
and run against a precast IDGel.TM. (made with Tris pH 8.3 as
buffering solution). After the electrophoretic run, the migration
speed for the migration front to reach the bottom of the gel, and
the resolution of a prestained molecular weight marker containing
10 bands weighing between 15 KDa to 175 KDa was measured. The
resolution of each of these bands has been compared to a control
IDGel.TM. ran with the standard Laemmli running buffer
(Tris-Glycine-SDS). The results we obtained are as follows in table
1.
TABLE-US-00001 TABLE I Gel size Zwitterion (migration length)
Running time Resolution AMPD 60 mm 45 min ++ AMPSO 60 mm 50 min +++
BES 60 mm 40 min ++(+) CAPS 60 mm 40 min ++ CAPSO 60 mm 60 min
(+++) CHES 60 mm 42 min +(+) EPPS (or HEPPS) 60 mm 45 min +++
Glygly 60 mm 45 min ++(+) POPSO 60 mm 50 min ++ TAPS 60 mm 35 min
+++ TES 60 mm 40 min +++ TGS* 60 mm 60 min ++ *Tris-Glycine-SDS =
baseline
[0094] The resolution is compared by measuring the thickness of
proteins bands of 30 KDa, 21 KDA, 15 KDA and 14.3 KDa after
migration in the test gel. A reduction of the band thickness by at
least 50% will be noted as a "+++" sign. Thicker protein bands,
indicative of lower resolution, is shown with less "+" signs. The
baseline is "++", which is the result obtained by a
Tris-Glycine-SDS gel, and a lesser number of "+" signs indicate
thicker bands than baseline, and therefore worst average
resolution.
[0095] Now referring to FIG. 2. The change in the band thickness
and resolution of four low molecular weight proteins was measured
FIG. 2 shows that the presence of EPPS (or HEPPS) in the buffer
helps improving the resolution by at least 50%. As well, the
complete migration of each of these gels was accelerated by 30%
using EPPS (or HEPPS).
Example 2
[0096] The same electrolyte solutions tested as running buffer
above can be used to prepare the acrylamide or agarose gel
composition and provide increased resolution, but not necessarily
separation speed.
TABLE-US-00002 TABLE 2 Gel size Zwitterion (migration length)
Running time Resolution.sup.1 EPPS (or HEPPS) 60 mm 50 min +++
Glygly 60 mm 50 min ++ TGS* 60 mm 60 min ++
Example 3
[0097] Some of these compounds have been identified to have a
positive impact on stabilizing the gel matrix and allowing an
extended storage, or more stable aging, providing longer shelf-life
at 4.degree. C. or room temperature. Now referring to FIG. 3. The
figure shows that gels kept at 4'C and tested every 30 days to
detect the impact of aging. The results show that after 360 days,
the gel migration is similar to a fresh 1-day old precast gel.
These results confirm the stability of the gel matrix when EPPS (or
HEPPS) is used as a buffering agent.
Example 4
[0098] The buffer of the present invention may also be used a
transfer buffer in applications such as the transfer of proteins
from acrylamide gels (Western transfer). Exemplary buffers were
prepared according to table 3, below.
TABLE-US-00003 TABLE 3 # Compound T0 T1 T2 T3 T4 Tris (g) 3 18 3 9
6 Glycine (g) 14.4 -- -- -- -- EPPS(or -- 12 2 6 4 HEPPS) (g)
H.sub.2O (ml) to 800 800 800 800 800 Methanol (ml) 200 200 200 200
200 T0 = Laemmli buffer with a glycine base.
[0099] Transfer of protein markers from a precast IDGel.TM. (made
with Tris pH 8.3 as buffering solution). The transfer was most
efficient with the recipe of buffer T4.
Example 5
[0100] Several buffers according to the present invention are
tested. The zwitterions tested are listed in table 4
TABLE-US-00004 TABLE 4 Testing of zwitterions of interest
Composants Symetrical Cathode Anode Tris (g) 6 6 1 EPPS(or 4 1 4
HEPPS) (g) Gly-Gly (g) 4 1 4 CAPSO (g) 4 1 4 BES (g) 4 1 4 Methanol
0% 10% 20% 0% 10% 20% 0% 10% 20% Total volume 1 litre 1 litre 1
litre
[0101] The transfer efficacy of the different tested zwitterions
are:
TABLE-US-00005 EPPS (HEPPS) 100% Gly-Gly 100% CAPSO 90% BES 80%
Example 6
[0102] Several buffers according to the present invention are
tested in an asymmetric configuration, as indicated in table 5.
TABLE-US-00006 TABLE 5 transfer of asymmetric buffer: Components
Cathode Anode Tris (g) 6 1 Gly-Gly (g) or EPPS 1 6 (or HEPPS)
Methanol 10% 10%
[0103] In each case, for the presented asymmetric arrangements with
Gly-gly or EPPS (or HEPPS) buffer, the transfer is 100% efficient
and occurs in 15 min of electrophoresis.
Example 7
[0104] The Tris/Gly-Gly buffer according to the present invention
(see Example 5) may be used for Western transfer without any
further modification. The buffer also supports an accelerated
separation/transfer: a current of 275 mA results in a complete
transfer in 15 minutes or less. Methanol is normally added to the
transfer buffer to slow the protein migration and normally permit a
more equal protein transfer between the high molecular weight
proteins and the low molecular weight. In standard protocol, it is
used as 10% or 20% of methanol. The efficacy of fast transfer with
electrolyte solutions according to the present invention containing
0% to 20% methanol is working equally well to 100% efficacy in
transfer under all these conditions.
Example 8
[0105] To perform semi-dry and/or wet transfer, the an electrolyte
solution according to the present invention may be used. The
solution containing EPPS (or HEPPS) is prepared as a 0.3.times.
solution: 6 g Tris, 4 g EPPS (or HEPPS) in a total volume of 1 L of
distilled water. The electrolyte solution may comprise between 0%
to 20% methanol, and preferably between 10 and 20% methanol to
improve transfer of large molecular weight proteins. The transfer
of sample from the gel at 275 mA is typically completed in 10 to 15
minutes.
Example 9
[0106] For use in electrophoresis transfer system such as the
eStain.TM., since the volume of electrolyte solution required is
small, two blotting papers are soaked in a 5.times. electrolyte
solution according to the present invention (90 g Tris, 60 g EPPS
(or HEPPS) and up to a 1 L total volume with water) and are placed
on each side of the gel from which the proteins are transferred.
The transfer is complete in 10 minutes at about 80 mA to about 300
mA, 100% of the sample is transferred to the PVDF membrane
used.
Example 10
Improved Buffer for Mops Based Gels
[0107] A protein electrophoresis running buffer that accelerates
the separation speed and increase resolution of MOPS based gels and
other gels. The buffer is based on the Tris-EPPS-SDS buffer in
combination with the ACES, MOPS, and PIPES zwitterions This buffer
will be advantageous on gels based on a different chemistry than
Laemmli's, for example MOPS based gels such as NuPage.TM. from
Invitrogen and EZ-Run.TM. from ThermoFisher, chemistry proprietary
to Amresco.
[0108] The basic recipe of the buffer is a combination of
Tris+SDS+EPPS+ACES+MOPS+PIPES, at a pH 8.5 or less and may
facultatively include one anionic surfactant to provide to the
buffer denaturing properties for protein analysis (e.g. SDS, sodium
dodecyl sulphate, Sodium lauryl sulfate (SLS), sodium laurilsulfate
or sodium, NaDS).
[0109] The proportions of this electrolyte solution are as
indicated in table 6.:
TABLE-US-00007 TABLE 6 Components of the composition mM % weight 1X
TRIS 49.9% 74.3 SDS 2.8% 3.5 EPPS 19.4% 27.7 ACES 9.7% 19.2 PIPES
1.7% 2.0 MOPS 16.6% 95.6
[0110] Under such proportion, the buffer has a pH of 7.4, which is
compatible with the MOPS based gels having a pH of 7.0.
Example 11
Improved Buffer for Mops Based Gels
[0111] This study was made using the liquid gel matrix EZ-Run.TM.
from ThermoFisher.TM., a product developed by Amresco.TM.. This
liquid gel matrix has the advantage to be stable over one year at
room temperature and allow to quickly cast electrophoresis gels.
The gel matrix comes with its specific running buffer, which has to
be used with it. The recommended running conditions are 150V.
[0112] The main problem related to this liquid gel matrix is that
it takes longer than a normal Laemmli gel or other precast gels
available on the market to make a normal gel separation. For this
reason, the EPPS based running buffer of the present invention is
adapted to this MOPS based gel matrix (see US Patent publication
No. 20060118418).
[0113] The performances of the new buffer are represented in the
following table. "Long gel"=10.times.10 cm and "Short
gel"=10.times.8.2 cm.
TABLE-US-00008 TABLE 7 Performance EZ-Run vs IDFast Univ Long gel
150 V EZRun 110 min EPPS mix 80 min 27% faster Long gel 200 V EZRun
80 min EPPS mix 50 min 38% faster Short gel 150 V EZRun 65 min EPPS
mix 45 min 31% faster Short gel 200 V EZRun 50 min EPPS mix pH 7.4
35 min 30% faster EPPS mix 2 pH 8 30 min 40% faster Also compatible
with Laemmli gels (short gels, 200 V) EZRun 50 min EPPS only buffer
35 min EPPS mix 29 min 17% faster EPPS mix pH 7.4 29 min
[0114] The gel prepared with the EZ-Run gel matrix and migrated
with the EZ-Run specific running buffer, when compared to the EPPS
based buffer of the present invention, as described in Example 9,
clearly shows an advantage in migration speed for the electrolyte
solution of the present invention without loss of resolution (See.
FIG. 6).
[0115] The pH of the electrolyte solution will also affect the
speed. The EZ-Run gel matrix has a pH of 7.0. The EZ-Run running
buffer has a pH of 8.0. The electrolyte solution of the present
invention (according to Example 9) has a pH of 7.4 once mixed, but
adjusting it to 8.0 with NaOH has an important impact on the
speed.
[0116] The electrolyte solution of the present invention can be
used alone, as well as in combination with the buffer specifically
designed for this gel matrix (EZ-Run running buffer in this
example). The proportion can change as desired, and shows
compatibility in the resolution and a proportional speed of
migration. Based on the results above, it appears that the EZ-Run
buffer slows down the migration.
Example 12
Improved Resolution and Gradient Effect of EPPS Based Gels
[0117] An electrolyte solution according to the present invention
is identified to have a positive impact on the resolution of the
protein bands separated on the gel. The electrolyte solution
improves both the definition (sharpness) and the separation of the
protein bands separated. Now referring to FIGS. 7A, B and C, three
polyacrylamide gels were prepared. Gel A is a precast IDGel.TM.
Tris-Glycine gel with 10% acrylamide; gel B is an IDGel.TM.
Tris-Glycine gel with a gradient of 4% to 20% acrylamide; gel C is
a gel containing 50 mM EPPS with 10% acrylamide, according to the
present invention. All gels were run in a 50 mM EPPS and 50 mM Tris
containing buffer (IDFast) according to the present invention. FIG.
7 shows that compared to the Tris-glycine gels, the resolution of
the EPPS gel is much improved. The band linked by a line
corresponds to 25 kDa.
[0118] For example the EPPS containing gel (FIG. 7C) is capable of
resolving proteins better than a Tris-glycine gel of identical
acrylamide percentage (compare FIGS. 7A and C, lanes 1 vs. 9, lanes
2 vs. 10, and lanes 3 vs. 8). It may be observed that the smaller
molecular weight proteins (e.g. 6 kDa in lanes 3 and 8. 10 kDa and
17 kDa in lanes 1 and 9 and 19 kDa and 15 kDa in lanes 2 and 10)
are well distanced and distinguishable on EPPS containing gel,
while they are poorly separated and fused with the dye front in the
Tris-glycine gel. Furthermore, higher molecular weight bands are
somewhat sharper.
[0119] Also, the EPPS containing gel (FIG. 7C) is also capable of
providing better resolution than a Tris-glycine gradient (4 to 20%
acrylamide) gel (FIG. 7B). The gradient gel of FIG. 7B provides
distinctly better resolution than that of the Tris-glycine 10% gel,
providing separation of the smaller molecular weight proteins
(compare lanes 1 and 5, 2 and 6, and 3 to 4 and 7). Nevertheless,
when compared to the gradient gel, the EPPS containing gel (compare
FIGS. 7B and C) provides for greater distance between the protein
bands across the entire range of molecular weight, especially for
the smaller molecular weight proteins. The EPPS containing gel
permits for small differences in molecular weight of protein to be
made (e.g. compare lanes 4-5 to lanes 8-9. Interestingly, the EPPS
containing gel appears to migrate in a similar "gradient-like"
manner that is more commonly observed in gradient gels such as in
FIG. 7B, this without the presence of a gradient in the EPPS
containing gel, which is of a fixed 10% acrylamide concentration.
Note that the dye migration front of the EPPS containing gel (FIG.
7C) has already exited the gel, while it remains present in the
other gels.
[0120] The embodiments and examples presented herein are
illustrative of the general nature of the subject matter claimed
and are not limiting. It will be understood by those skilled in the
art how these embodiments can be readily modified and/or adapted
for various applications and in various ways without departing from
the spirit and scope of the subject matter disclosed claimed. The
claims hereof are to be understood to include without limitation
all alternative embodiments and equivalents of the subject matter
hereof. Phrases, words and terms employed herein are illustrative
and are not limiting. Where permissible by law, all references
cited herein are incorporated by reference in their entirety. It
will be appreciated that any aspects of the different embodiments
disclosed herein may be combined in a range of possible alternative
embodiments, and alternative combinations of features, all of which
varied combinations of features are to be understood to form a part
of the subject matter claimed.
* * * * *