Electrophoresis gels

Details Electrophoresis gels Electrophoresis gels

2001-02-16

2004-05-11

Noguerola, Alex (Department: 1753)

Chemistry: electrical and wave energy

Apparatus

Electrophoretic or electro-osmotic apparatus

C204S616000, C204S470000

Reexamination Certificate

active

06733647

ABSTRACT:

TECHNICAL FIELD
This invention relates to the field of gel electrophoresis, particularly to pre-cast polyacrylamide gels having an extended shelf-life.
BACKGROUND ART
Gel electrophoresis is an important analytical and preparative separation technique in which charged molecules are separated under the influence of an electric field with a gel being used as the support matrix. This technique is particularly suitable for the separation of biological macromolecules. The gels commonly used in this technique are composed of polyacrylamide or agarose. Polyacrylamide gels are used particularly for the separation of biomolecules such as proteins, peptides, DNA, RNA, lipids, charged carbohydrates and the like, either naturally occurring or synthetic, in which the acrylamide is used in slab form being pre-cast prior to use. Traditionally, polyacrylamide gels have been prepared individually prior to use by polymerising an acrylamide/cross-linker solution in a gel-casting cassette to form a slab. Following electrophoresis, the gels are removed from the cassette, and the biomolecules are stained and/or transferred from the gel to another medium so that the separated biomolecules may be visualised, identified, recovered or quantified. Conventional polyacrylamide gels have the disadvantage of being relatively unstable and have a limited shelf-life.
As gels are often prepared on an individual basis prior to use, there can be variations between gels that have been cast separately such that direct comparisons between separations using different gels are not reliable. Furthermore, the monomer components in polyacrylamide gels are relatively toxic and continued preparation of gels increases the potential of exposure of these toxic monomers to the operator. There has now been a move to the commercial preparation of pre-cast gels under controlled conditions providing consistent and stable characteristics between batches of gels. Unfortunately, most pre-cast commercial gels still have the problem of limited shelf-life and must be used within a relatively short period of time to ensure accurate and reliable separations.
Currently, a major limitation in the production and sale of pre-cast polyacrylamide electrophoresis gels is the relatively short shelf-life, usually up to about three months. This is thought to be due to the hydrolysis of the amide groups in polyacrylamide to the carboxylic acid derivative in alkaline conditions [Geisthardt & Kruppa, Polyacrylamide Gel Electrophoresis: Reaction of Acrylamide at Alkaline pH with Buffer Components and Proteins
Anal. Biochem.
160, 184-191 (1987)]. This hydrolysis is manifested in the gels as a loss of resolution of separated molecules, change in the migration distances of the separated molecules and reduced intensity of protein staining.
Typically, gels are prepared using alkaline buffers and run under alkaline conditions, usually around pH 8.9. A buffer system using Tris(hydroxymethyl)aminomethane and hydrochloric acid (Tris-HCl) developed by Laemmli [Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4
Nature
227, 680-686 (1970)], is a typical choice for “standard’ polyacrylamide gels in denaturing conditions. Although loss of stability in polyacrylamide gels occurs faster in alkaline conditions, this was thought to be unavoidable in standard gels. In principle, if the pH of the buffer in which the polymer network is formed could be lowered to around neutral, then the hydrolysis of the gel should be greatly reduced and so the gels should remain stable and useful for a longer period of time. Unfortunately, it has been difficult to find inexpensive chemical systems with an effective buffering capacity around neutral pH that are compatible with a polyacrylamide medium and suitable for gel electrophoresis and which do not have any effects that would cause a loss of stability through other interactions.
The most common buffer system used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is that published by Ormstein [Disc Electrophoresis,
1
, Background and Theory
Ann. New York Acad. Sci.
121, 321-349 (1964)] and modified by Laemmli. This system uses a discontinuous electrophoresis system composed of a ‘stacking’ gel with a Tris concentration of 0.125 M at pH 6.8 and a ‘resolving’ gel with a Tris concentration of 0.375 M at pH 8.8. The change in pH causes the proteins in the gel to ‘stack’ or concentrate into a fine line in the lower pH gel and then ‘resolve’ or spread out in the higher pH gel. The stacking gel is a short zone and the resolving gel is a longer zone in the gel. The other common Tris-HCl system is that of Schagger and von Jagow [Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa
Anal. Biochem
166, 368-379 (1987)] who used a stacking gel of 0.75 M Tris at pH 8.45 and a resolving gel of 0.9 M at pH 8.45. Some reports have been made of the use of the Tris-HCl system below these pH values. Reisfield and Williams [Disc Electrophoresis in Polyacrylamide Gels: Extension to New Conditions of pH and Buffer
Ann. N.Y. Acad. Sci.
121, 373-381 (1964)] have used Tris-HCl at a pH of 7.5, but their Tris concentration was very low (around 0.08 M) and their electrode buffer was Tris-diethylbarbituric acid. The present inventors have found that using Tris at these very low concentrations and at low pHs in more common electrophoresis of buffers leads to a distortion of bands and lack of resolution. King et al [Electrophoretic Conditions for High Resolution Citrus Isozymes in Polyacrylamide Gel Electrophoresis
Electrophoresis
16, 32-38 (1995)] have used traditional concentrations of Tris (0.375 M) at pH 7.5. To reach the lower pH levels at this high concentration of Tris involves the addition of a large amount of hydrochloric acid leading to high conductivity in the gel. When the conductivity of the gel is high, the gels run very slowly in traditional electrode buffers.
A number of different gel buffer systems have been proposed for use at or around neutral pH that do not involve the use of Tris-HCl. The most common buffer system at neutral pH is the phosphate system of Shapiro [Biochem.
Biophys. Res. Commun.
28, 815 (1967)] modified by Weber and Osborn [The Reliability of Molecular Weight Determinations by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
J. Biol. Chem.
244(16), 4406-4412 (1969)]. Other buffer systems include Bis-Tris-HCl [Moos et al Reproducible High Yield Sequencing of Proteins Electrophoretically Separated and Transferred to an Inert Support
J. Biol. Chem.
263(13), 6005-6008 (1988)], Tris-Acetate [Patton et al Tris-Tricine and Tris-Borate Buffer Systems Provide Better Estimates of Human Mesothelial Cell Intermediate Filament Protein Molecular Weights than the Standard Tris-Glycine System Anal. Biochem. 197, 25-33 (1991)], glycylglycine-NaOH [Hoffmann & Chalkley A Neutral pH Acrylamide Gel Electrophoretic System for Histones and Other Basic Proteins
Anal. Biochem.
76, 539-546 (1976)], 1,4 piperazine-bis-(ethane sulfonic acid) (PIPES) —Na
3
PO
4
[Davis & Gregerman Separation of Thyroxin (T
4
)-Binding Proteins of Human Serum in Polyacrylamide Gel at pH 7.4. I. Effect of pH on Distribution of Tracer Quantities of T
4
J. Endocr.
30, 237-245 (1970)], 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid (HEPES)/2-(N-morpholino)ethanesulphonic acid (MES)/3-(N-morpholino)propane sulphonic acid (MOPS)/Bicine-NaOH [12], MOPS-KOH [Thomas & Hodes A New Discontinuous Buffer System for the Electrophoresis of Cationic Proteins at Near-Neutral pH
Anal. Biochem.
118, 194-196 (1981)] and Histidine-HCl/Tris-Citrate [King et al Electrophoretic Conditions for High Resolution Citrus Isozymes in Polyacrylamide Gel Electrophoresis
Electrophoresis
16, 32-38 (1995)].
The buffering capacity of Tris-HCl is reduced at neutral pH and so commercially available pre-cast

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