WO2000067009A1 - Tampons melanges dans des systemes d'electrophorese en gel - Google Patents

Tampons melanges dans des systemes d'electrophorese en gel Download PDF

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WO2000067009A1
WO2000067009A1 PCT/US2000/011474 US0011474W WO0067009A1 WO 2000067009 A1 WO2000067009 A1 WO 2000067009A1 US 0011474 W US0011474 W US 0011474W WO 0067009 A1 WO0067009 A1 WO 0067009A1
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separation
range
electrophoresis
polymeric material
biomolecules
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PCT/US2000/011474
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English (en)
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Lih-Bin Shih
Patricia Vilalta
Mark Williams
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Applied Hydrogel Technology Corporation
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Priority to AU46758/00A priority Critical patent/AU4675800A/en
Publication of WO2000067009A1 publication Critical patent/WO2000067009A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • the present invention relates to gel electrophoresis, and particularly to improved methods for the separation of biological materials using gel electrophoresis.
  • Gel electrophoresis is a technique that is often carried out for the analysis and purification of biological molecules such as peptides, proteins, nucleic acids, carbohydrates, and others. Electrophoresis is based on the principle that charged molecules will migrate when placed in an electric field, with the rate of migration dependent upon, among other things, the molecule's charge density (charge per unit of mass). Gel electrophoresis uses a gel, made of natural or synthetic polymers, typically manufactured in a slab orientation or alternatively in a tube shape, to provide a support for the molecules so that they can be analyzed at the completion of separation, and to provide additional sieving characteristics.
  • Gels for electrophoresis can be considered to consist of random networks of filaments, cross-linked by covalent, ionic, hydrogen or other molecular bonds, to form a three-dimensional "sponge".
  • the pores form a molecular sieve that retards the mobility of molecules migrating in the imposed electric field.
  • the size of the pores is determined by the concentration of filaments (normally described as the percentage of solid in the gel) and the degree of cross-linking. For two molecules with equal charge density, their migration through the gel will be determined by their size.
  • the rate of migration is inversely proportional to the log of the protein molecular weight over a limited range of molecular weights, so that smaller molecules migrate at a faster rate than those of larger size.
  • Gel electrophoresis is typically performed by placing a complex mixture of molecules to be separated and analyzed or purified at one position in the gel by means of a well formed in the gel or by means of layering at one end of a tube.
  • the mixture usually contains a visible dye that migrates at a known rate in the system.
  • the gel is brought into contact with an electrolyte buffer solution that is in electrical contact with a power source, and a defined electrical field is applied to the gel.
  • the molecules in the sample migrate toward the appropriate electrode, each at an individual rate until the marker dye reaches a predetermined position in the gel.
  • the electrical field is then removed and the separated molecules are visualized by one or more methods well known in the field as bands corresponding in width to the width of the sample well. The size, relative concentration and other information on the molecules in the sample can then be determined.
  • nucleic acids the charge density of each molecule is approximately equal, since each individual nucleotide has one charged moiety. Care is taken to remove any possible tertiary structure from RNA and DNA, and any secondary structure from RNA and DNA for determinations based on specific sequence characteristics, typically by using a combination of heat and chemical denaturation. As a result, nucleic acids are typically separated based on their primary sequence length.
  • the extent of molecular sieving is thought to depend on how closely the gel pore size approximates the size of the migrating particle.
  • the pore size of agarose gels is sufficiently large that molecular sieving of most protein molecules is minimal and separation of proteins is based mainly on charge density.
  • polyacrylamide gels can have pores that more closely approximate the size of protein molecules and so contribute to the molecular sieving effect.
  • Polyacrylamide has the further advantage of being a synthetic polymer which can be prepared in highly purified form.
  • Polymeric materials consisting of block copolymers of partially hydrolyzed acrylonitrile have also been demonstrated to be of utility in the separations of biological materials (US Patent 5,888,365).
  • Gels are typically saturated with an electrolyte solution that provides for efficient electrical conductivity.
  • the rate of biomolecule migration in gel electrophoresis is also affected by the concentration and type of ions present in the electrolyte solution.
  • the concentration of ions in the electrolyte solution must be high enough that the counter- ions keeping the biological molecule in solution are not separated from the biomolecule (which is itself a large ionic species), in which case the force attracting the large ion to the small counter-ions can become stronger than the electromotive force of the applied electric field.
  • Electrolyte concentrations that are too great lead to degradation of molecule band resolution.
  • the pH of the electrolyte also plays a role in maintaining electrophoretic separation.
  • the electrolyte solution also contains a buffering system, typically based upon tris(hydroxy-methyl)aminomethane (Tris).
  • sample buffer is used to prepare the biological materials for separation under a given set of electrophoretic conditions.
  • electrode buffer is used as a conducting medium, among other functions, to conduct the electrical potential from the electrode to the gel, which causes the charged biological molecules to migrate and separate in the gel.
  • gel buffer which impregnates the gel media so as to cause the biological molecules to migrate and resolve themselves according to their size and charge under electrophoretic conditions.
  • Gel buffers are often chosen so that the media has a suitable pH value for biological molecules to carry desired charges under a given set of electrophoretic conditions.
  • the buffer type and ionic strength are also factors of consideration for achieving the optimal results of (a) controlling the pore size of the gel for sieving and (b) controlling and balancing the heat generation and the speed of separation.
  • Tris-HCl Tris-Borate
  • Tris-acetate Tris-acetate
  • the electrode buffer often consists of a Tris-glycine system, or the like.
  • electrolyte components such as SDS in the buffer system, but it has been very uncommon in the past for organic agents such as ethanol, methanol, acetonitrile, and the like to be added to gel buffer systems for use in electrophoretic applications.
  • polyacrylamide is a very hydrophilic material whose polymerization is adversely affected by some agents that interfere with the free-radical initiated polymerization of polyacrylamide, resulting in lack of gelation, opaque gels, gels with reduced separation capacity, and other problems.
  • some organic agents have been used in combination with polyacrylamide and other polymer gels, most notably sulfolane, dimethyl formamide (DMF), urea, and phenol. These agents have been added to polyacrylamide gels with the intent of denaturing or solubilizing otherwise insoluble, hydrophobic biomolecules to make gel electrophoresis possible.
  • Electrophoresis 1992, 13:824-831 reports the use of TMU, DMF, formamide, methanol, ethanol, DMSO, sulfolane, n-methylacetamide, N- methylpyrrolidinone and tetrahydrofuran to reswell dried polyethyleneglycol methacrylate/polyacrylamide gels, and suggests that the agents are compatible with this copolymer system.
  • This paper describes experiments designed to allow for separation of extremely hydrophobic proteins and notes the gels studied "as a whole have less rapid protein migration and poorer resolution" (p 829).
  • Electrophoresis 1994 15:195-199 describes poly(ethylene glycol) methacrylate- acrylamide copolymer media developed to enable electrophoresis of hydrophobic proteins.
  • the authors describe increasing the speed of electrophoresis in this media by addition of 25% methanol or 30% DMF, which act as anti-freeze and allow for running gels at temperatures beginning at -20° C.
  • the authors claim their system provides optimal results when the gel temperature is held at 12° C or below. Using gels containing antifreeze and starting runs at -20° C, the authors were able to apply much higher current while staying below the 12° C limit, resulting in 2 to 6-fold faster run times.
  • non-gel materials As yet another example of separation systems designed for use with organic agents, non-gel materials (sponges) have been described that are compatible with organic agents (DMSO) and which are appropriate for electrophoresis of highly hydrophobic proteins [Electrophoresis 1994 15:187-194]. See also US Patent 5,637,202.
  • invention methods and materials can serve to increase the rate of migration of biologic molecules in gel electrophoresis applications. As a result, the length of time required to complete a given electrophoresis run is decreased, allowing for higher data throughput in a given time period.
  • the present invention has positive implications for increasing the efficiency of high-throughput biological molecule screening and characterization.
  • Electrophoresis gels contemplated for use in the practice of the present invention provide advantages over gels currently employed in the art. Decreases in separation times allow for additional gels to be run in a given time period, increasing efficiency in life science laboratories. Inclusion of organic agents allows for efficient separation of proteins with strong hydrophobic character compared to gels without added organic buffer.
  • inventions comprise: a) applying a mixture of biomolecules to a separation medium comprising a gellable polymeric material and a mixed medium, wherein said mixed medium comprises an aqueous component and one or more organic agents, b) applying an electric field to said separation medium, for a time sufficient to cause the electrophoretic separation of said biomolecules, according to the size and relative charge of said biomolecules.
  • the aqueous component of the mixed medium contemplated for use in the practice of the present invention will comprise electrolytes in sufficient concentration, and combination, and having a suitable pH for conducting electrophoresis.
  • electrolytes will depend on the gellable polymeric material being used as well as the nature of the biomolecules being separated. Those of skill in the art can readily determine the appropriate type and amount of electrolytes to be employed.
  • the aqueous electrolyte-containing component contemplated for use in the practice of the present invention comprises buffered media having a pH in the range of about 2 up to 12, such aqueous components include aqueous surfactant-containing mixtures, aqueous solutions containing salt or other electrolytes, and the like.
  • aqueous components include aqueous surfactant-containing mixtures, aqueous solutions containing salt or other electrolytes, and the like.
  • organic agent to the separation media allows for higher running voltages at a given temperature without damage to the gel from heating. Accordingly, invention methods may be carried out at any temperature provided that the electric field applied to the gel does not damage the separation medium to an extent that prevents accurate electrophoresis. As will be understood by those of skill in the art, the accuracy of electrophoresis can be gauged by running appropriate molecular weight standards, or the like.
  • application of electric field to the separation medium is conducted at or below room temperature. In another embodiment of the present invention, application of electric field to the separation medium is conducted at or above room temperature. In a presently preferred embodiment of the present invention, application of electric field to the separation medium is at room temperature. In a presently preferred embodiment, electrophoresis is conducted at a temperature range from about -20°C to room temperature (i.e., about 22°C).
  • Electrophoresis according to invention methods can be conducted with the application of any suitable electric field. Accordingly, in one embodiment of the present invention, the applied electric field is in the range of about 125 V to 5000V. In another embodiment of the present invention, the applied electric field is in the range of about 1000V to 2000 V. In still another embodiment of the present invention, the applied electric field is in the range of about 2000V to 5000V. In still another embodiment of the present invention, the applied electric field is in the range of about 250V to 1000V. In a presently preferred embodiment of the present invention, the applied electric field is in the range of about 350V to 450V.
  • organic agent means any organic compound which, when added to the separation medium and mixed medium described herein, will provide for faster electrophoresis. Without wanting to be bound by theory, this effect is believed to be a result of a change in conductivity in the electrophoresis system due to changed viscosity and dielectric constant of a buffer containing organic agents. Molecular species are able to migrate faster in a given electrical field under the viscosity and dielectric constant conditions formed by the inclusion of organic agents in electrophoresis buffers.
  • Suitable organic agents contemplated for use in the practice of the present invention include those that are at least 5% soluble in aqueous medium, such as select alcohols, ketones, nitriles, polar aprotic solvents, ethers, carbamides, and the like.
  • Alcohols contemplated for use in the practice of the present invention include those that are at least 5% soluble in aqueous medium, such as methanol, ethanol, isopropanol, phenol, m-cresol, and the like.
  • Ketones contemplated for use in the practice of the present invention include those that are at least 5% soluble in aqueous medium, such as acetone, methyl ethyl ketone, ethyl ethyl ketone, acetophenone, and the like.
  • Nitriles contemplated for use in the practice of the present invention include those that are at least 5% soluble in aqueous medium, such as acetonitrile, glutaronitrile, propionitrile, or the like.
  • Ethers contemplated for use in the practice of the present invention include those that are at least 5% soluble in aqueous medium, such as tetrahydrofuran, dioxane, dimethyl ether, or the like.
  • Carbamides contemplated for use in the practice of the present invention include those that are at least 5% soluble in aqueous medium, such as urea, tetramethyl urea (TMU), or the like.
  • aqueous medium such as urea, tetramethyl urea (TMU), or the like.
  • Polar aprotic solvents contemplated for use in the practice of the present invention include dimethyl formamide, dimethyl sulfoxide, chloral hydrate, formamide, benzaldehyde, ethyl acetate, N-methylacetamide, N-methylpyrrolidinone, and the like.
  • the mixed medium comprises in the range of about 20 to about 99.5 wt% of aqueous component. In another embodiment of the present invention the mixed medium comprises in the range of about 60 to about 99 wt% of aqueous component. In yet another embodiment of the present invention, the mixed medium comprises in the range of about 80 to about 99 wt% of aqueous component. In a presently preferred embodiment, the mixed medium comprises about 95 wt% of aqueous component.
  • the amount of organic agent in the mixed medium contemplated for use in the practice of the present invention will also vary according to the particular organic agent and gellable polymeric material employed.
  • the mixed medium contains about 1-30 wt% of organic agent(s).
  • the mixed medium comprises in the range of about 3 to about 20 wt% of organic agent(s).
  • the mixed medium comprises in the range of about 4 to about 10 wt% of organic agent(s).
  • the mixed medium comprises about 5 wt% of organic agent(s).
  • methods for the rapid separation of biomolecules by size and relative charge comprising: a) applying a mixture of biomolecules to a modified separation medium comprising a gellable polymeric material and a suitable electrophoresis buffer, b) applying an electric field to said modified separation medium, for a time sufficient to cause the electrophoretic separation of said biomolecules, according to the size and relative charge of said biomolecules, wherein the viscosity and/or dielectric constant of said modified separation medium is selected such that biomolecules separate during electrophoresis therein at a more rapid rate than in unmodified separation medium.
  • Suitable dielectric constants for separation media which provide more rapid separation of biomolecules typically fall in the range of about 5-75, and preferably in the range of about 10-40.
  • Suitable viscosities for separation media which provide more rapid separation of biomolecules typically fall in the range of about 0.2 to 2.0 mPa s at 25°C.
  • the separation medium further comprises a denaturing agent.
  • a denaturing agent Any appropriate denaturing agent can be employed, including sodium dodecyl sulfate, urea, formamide, sulfolane, ⁇ -mercaptoethanol, dithiothreitol (DTT), methyl mercury, or the like.
  • Biomaterial contemplated for treatment in accordance with invention separation methods include proteins, polypeptides, nucleic acids, oligonucleotides, carbohydrates, oligosaccharides, lipids, glycolipids, as well as other charged polymine materials (both natural and synthetic), and the like.
  • polypeptides refer to compounds containing two or more amino acids linked covalently through peptide bonds. Polypeptides of three or more amino acids are sometimes referred to as oligopeptides.
  • proteins refer to macromolecules made up of one or more chains of amino acids covalently joined through peptide bonds. Proteins can vary greatly in molecular weight, from a few thousand to several million daltons.
  • nucleic acids refer to either of two major types of macromolecule (i.e., DNA or RNA) formed by polymerization of nucleotides.
  • DNA and RNA exist in many forms, all of which are contemplated for use in the practice of the present invention.
  • oligonucleotides refer to relatively short chain nucleic acids, i.e., from 2 to about 1000 bases in length.
  • Carbohydrates refer to sugar-based compounds containing carbon, hydrogen and oxygen with the general formula C x (H O) y . Carbohydrates can be divided into various sub-groups, i.e., monosaccharides, disaccharides, oligosaccharides or polysaccharides, depending on the degree of polymerization of the basic sugar units. As employed herein, “oligosaccharides” refer to carbohydrates containing a plurality of monosaccharides. As employed herein, “lipids” refer to those compounds found in living organisms which are not carbohydrates, proteins or polynucleic acids.
  • Lipids tend to be soluble in organic agents and insoluble in water, and include fats, waxes, phospholipids, glycolipids, steroids, te ⁇ enes and a number of different types of pigments.
  • the major group of lipids contains those compounds whose structure is characterized by the presence of fatty acid moieties (acyl lipids). These include neutral lipids (glycerides and waxes) and polar lipids (phospholipids and glycolipids).
  • acyl lipids fatty acid moieties
  • lipids include neutral lipids (glycerides and waxes) and polar lipids (phospholipids and glycolipids).
  • glycolipids refer to lipids that contain one or more carbohydrate moieties. These lipids include the cerebrosides and ganghosides in animals and the galactosyl diglycerides and sulpholipids in plants.
  • the lipid portion is usually glycerol phosphate, gly
  • the gellable polymeric material contemplated for use in the practice of the present invention may comprise thermally crosslinked gels of different monomer and crosslinker combinations selected for their suitability in separation of particular molecular species.
  • Exemplary thermally crosslinked gellable polymeric materials contemplated for use in the practice of the present invention include polyacrylamide, substituted acrylamide (including, for example, N-substituted polyacrylamide, and the like), poly-N-acryloyl-tris, acrylomo ⁇ holine, HYDROLINKTM, acrylic and methacrylic esters (e.g., poly(ethylene glycol) methacrylate-acrylamide copolymer, polyhydroxy methacrylate, poly(ethylene glycol) methacrylate, and the like), poly(N-acylalkylenimine), N-vinyl pyrrolidone-based polymers, methacrylic acid-based polymers (e.g., glycerol methacrylate-based polymers,
  • the gellable polymeric material contemplated for use in the practice of the present invention may comprise gels of natural or synthetic polymers which form gels through chain-entanglement processes.
  • Such gellable polymeric materials include agarose (natural material) and the synthetic hydrogel materials described in United States Patent No. 5,888,365 (inco ⁇ orated herein by reference in its entirety; referred to hereinafter as "AHT hydrogel"), where the gel network formation is based on chain entanglement of the hydrophobic domains of nitrile groups in a polyacrylonitrile-polyacrylamide copolymer, greater hydrophobic character is present in gels, by virtue of the presence of the nitrile groups, than that of the conventional polyacrylamide gel material used in electrophoresis. Because of the hydrophobicity of the AHT hydrogel material, the electrophoretic process can take place at a far higher voltage (and thus speed) while the heat generated by the process can be dissipated to tolerable levels by air cooling or a simple water bath.
  • electrophoresis matrices comprising chemically modified or differentially crosslinked acrylamide and other polymers have been developed for separation of biomolecules and are reviewed in Electrophoresis 1995 16:1815-1829, inco ⁇ orated herein by reference in its entirety. Many of these are compatible with organic agents and are contemplated for use in the practice of the present invention.
  • Additional gellable polymeric materials contemplated for use in the practice of the present invention include electrophoresis sponges such as those comprising polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidine difluoride, polynitrile, polystyrene, or the like, or two or more thereof.
  • gellable polymeric materials One key property of the foregoing gellable polymeric materials is the ability to uptake a large weight percentage of aqueous medium.
  • suitable materials will be sufficiently hydrophilic so as to allow the desired uptake level of aqueous medium.
  • gellable polymeric materials suitable for use in the practice of the present invention are those having sufficient strength, in the presence of aqueous media, to retain the structural integrity thereof.
  • Such structural integrity can be imparted by chemical linkage (e.g., covalent crosslinking, or ionic crosslinking), physical interaction (e.g., hydrogen bonding, hydrophobic interactions (such as the presence of crystalline domains), physical entanglement of polymer chains, dipolar forces, etc.), and the like.
  • chemical linkage e.g., covalent crosslinking, or ionic crosslinking
  • physical interaction e.g., hydrogen bonding, hydrophobic interactions (such as the presence of crystalline domains), physical entanglement of polymer chains, dipolar forces, etc.
  • dipolar forces make a significant contribution to the structural integrity of the gellable polymeric material
  • the pore size of the gel can be varied by appropriate modification of the chemical structure of the polymer, as well as manipulation of the electrolyte conditions (i.e., ionic strength, buffer type and pH).
  • Gellable polymeric materials suitable for use in the practice of the present invention can be either ionizable or non-ionizable, so long as they meet the criteria set forth herein.
  • Crosslinking agents when employed, are bifunctional compounds which serve to bridge two different polymer chains. Commonly used crosslinking agents are ⁇ , ⁇ - diolefins, which are inco ⁇ orated into the forming polymer by free radical polymerization. The degree of crosslinking imparted to the resin impacts the pore size achievable by the resulting resin. When chemical crosslinking agents are not used for the preparation of gellable polymeric material, gel pore size can be controlled by controlling the extent the gellable polymeric material is capable of chain entanglement and cohesive dipolar forces, and by controlling the electrolyte conditions (e.g., ionic strength, pH and buffer type) employed for the separation process.
  • electrolyte conditions e.g., ionic strength, pH and buffer type
  • the gellable polymeric material employed in the practice of the present invention forms a hydrogel, based at least in part, upon cohesive dipolar forces, the gel pore size can be varied by appropriate manipulation of the electrolyte conditions (e.g., ionic strength, buffer type and pH).
  • the separation medium is comprised of gellable polymeric materials characterized as thermally crosslinked gels (e.g., polyacrylamide), said gels containing in the range of about 4% to about 30% total monomer (generally expressed as %T), said gels formed by crosslinking polyacrylamide strands with crosslinking agent (e.g., bis-acrylamide) in the range of about 2.5% to about 10% (generally known as %C).
  • thermally crosslinked gels e.g., polyacrylamide
  • %T total monomer
  • crosslinking agent e.g., bis-acrylamide
  • Addition of an organic agent in the gel buffer of the gels to achieve the benefits of enhanced separation speed has not previously been practiced. However, it has su ⁇ risingly been found that addition of about 1-30%, and more preferably 5-10% of organic agent to traditional polyacrylamide gel media is quite beneficial, presumably as a result of decreased viscosity and dielectric constant of the modified media.
  • thermally crosslinked gels are equilibrated in gel buffer containing about 1-30% and more preferably about 5-10% of organic agent rather than carrying out polymerization in the presence of organic agent, in order to speed the polymerization step while retaining the beneficial aspects of inclusion of organic agents in the gel buffer.
  • the ratio of gellable polymeric material to mixed medium can vary widely depending on the intended use, the size and nature of biomolecules being separated, and the like.
  • separation media for use in an electrophoresis system, wherein said separation media comprises a gellable polymeric material and a mixed medium, and wherein said mixed medium comprises an aqueous component and one or more organic agents. All components in the amounts specified herein are suitable for use in the practice of this embodiment of the invention.
  • invention articles useful for the separation of biological materials.
  • Invention articles comprise a support having deposited thereon a layer of about 0.01-5 mm of a separation medium comprising a gellable polymeric material containing aqueous medium with added organic buffer absorbed therein, wherein said gellable polymeric material is characterized as described herein.
  • articles comprising a support structure containing therein separation medium comprising gellable polymeric material containing aqueous medium with added organic buffer absorbed therein, wherein said gellable polymeric material is characterized as described herein.
  • Support materials contemplated for use in preparation of invention articles include glass plates, plastic sheets, and the like.
  • gellable polymeric material can be inco ⁇ orated into support structures such as columns, glass tubing, capillary tubing, glass cells, and the like.
  • Suitable support structures can be constructed of a variety of materials, as can be readily determined by those of skill in the art (e.g., glass, plastic, and the like).
  • the gel buffer for AHT hydrogels comprises Tris (tris (hydroxy methyl) aminomethane)-borate buffer and 0.1% SDS with or without an organic agent such as 10% acetonitrile.
  • the running buffers contain Tris-glycine buffer and 0.1% SDS.
  • polyacrylamide (Laemmli, 1970 Nature, 227, 680-686) gel buffer typically consists of 0.375 M Tris-HCl, pH 8.8, in the separating gel.
  • the stacking gel consists of Tris-HCl, pH 6.8.
  • the anode and cathode polyacrylamide running buffers Tris-glycine buffer and 0.1 % SDS.
  • a series of AHT hydrogels that contained agents of decreasing dielectric constants were cast. Each gel was equilibrated in a buffer of appropriate composition for electrophoresis that included 5-20% of added organic agent such as acetonitrile, ethanol, acetone, or ethyl acetate. The gels were run without a stacking component. Gels were loaded with molecular weight standards (SIGMAMARKERTM) prepared according to the manufacturer's instructions in 2x Laemmli loading buffer and electrophoresis was performed in an electrophoresis cell made to accommodate the hydrogels. Electrophoresis was performed by applying a electric charge limited to 450 volts and 80 milliamperes to the apparatus. The electrophoresis was terminated when the leading front of bromophenol blue marker dye had migrated to a position 55 mm from the sample well.
  • SIGMAMARKERTM molecular weight standards
  • the time required to complete electrophoresis was recorded, and the gel was processed for staining with Coomassie Brilliant Blue stain.
  • the distance (in mm) traveled by the protein markers from the sample well was measured following completion of the Coomassie Brilliant Blue staining procedure.
  • the rate of electrophoretic separation (mm/min) was determined for each gel condition. In each case, gels with higher concentration of organic agent had a faster rate of separation than those with lower concentrations. For all gels with concentrations of organic agent at 10% or higher, the rate of separation was increased by 5 to 10% .
  • a series of polyacrylamide gels that contained agents of decreasing dielectric constants were cast. Each 5 ml gel contained 2.5 ml of 30% monomer acrylamide/bisacrylamide (29:1; 3.3% crosslinking), 0.375 M TrisHCl, pH 8.8, 0.1% SDS and 5-10% of an organic agent such as acetonitrile, ethanol, acetone, or ethyl acetate. The gels were run without a stacking component. Gels were loaded with molecular weight standards (SIGMAMARKERTM) prepared according to the manufacturer's instructions in 2x Laemmli loading buffer and electrophoresis was performed in a BioRad Mini-PROTEAN IITM apparatus.
  • SIGMAMARKERTM molecular weight standards
  • the electrode buffer used was 0.025 M Tris, 0.192 M glycine, 0.1% SDS, with 125 ml in the cathodic end of the apparatus and 250 ml in the anodic end.
  • the electrophoresis was performed by applying a constant 125 volts to the apparatus, and was continued until the leading front of bromophenol blue marker dye reached a point 5 mm from the bottom of the gel.
  • the time required to complete electrophoresis was recorded, and the gel was processed for staining with Coomassie Brilliant Blue stain according to well-known protocols.
  • the distance (in mm) traveled by the protein markers from the sample well was measured following completion of the Coomassie Brilliant Blue staining procedure.
  • the rate of electrophoretic separation (mm/min) was determined for each gel condition. Gels made with acetonitrile, acetone or ethyl acetate demonstrated increased rate of separation when compared with gels run in the absence of organic agent.
  • the rate of electrophoresis in a polyacrylamide gel that contained 5% acetonitrile was compared to a standard polyacrylamide gel with no added organic agent.
  • Each 5 ml gel contained 2.5 ml of 30% monomer acrylamide/bisacrylamide (29:1; 3.3% crosslinking), 0.375 M TrisHCl, pH 8.8, and 0.1% SDS.
  • the gels were run without a stacking component. Gels were loaded with molecular weight standards (SIGMAMARKERTM) prepared according to the manufacturer's instructions in 2x Laemmli loading buffer and electrophoresis was performed in a BioRad Mini- PROTEAN IITM apparatus.
  • SIGMAMARKERTM molecular weight standards
  • the electrode buffer used was 0.025 M Tris, 0.192 M glycine, 0.1% SDS, with 125 ml in the cathodic end of the apparatus and 250 ml in the anodic.
  • the electrophoresis was performed by applying a constant 125 volts to the apparatus for 60 minutes, at which time the gel was processed for staining with Coomassie Brilliant Blue stain according to well-known protocols.
  • the distance (in mm) traveled by the protein markers from the sample well was measured following completion of the Coomassie Brilliant Blue staining procedure.
  • the rate of electrophoretic separation (mm/min) was determined for each gel condition.
  • the gel made with acetonitrile demonstrated an increased rate of separation as compared to a standard polyacrylamide gel with no added organic agent.

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Abstract

L'invention concerne des procédés pour la séparation de matières biologiques dans lesquels on augmente la vitesse de séparation des matières biologiques en ajoutant une petite quantité d'agent organique au tampon d'électrophorèse. Ainsi, selon l'invention, on peut séparer et analyser des matières biologiques par électrophorèse en gel plus rapidement qu'avec les procédés antérieurs.
PCT/US2000/011474 1999-04-30 2000-04-28 Tampons melanges dans des systemes d'electrophorese en gel WO2000067009A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1167962A1 (fr) * 1999-12-02 2002-01-02 Hymo Corporation Gels de polyacrylamide premoules pour l'electrophorese, procede de production de ces derniers et procede d'electrophorese dans lequel on utilise ces gels
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WO2018217816A1 (fr) * 2017-05-22 2018-11-29 Integenx Inc. Compositions, procédés, kits et dispositifs d'analyse moléculaire
WO2018217789A3 (fr) * 2017-05-22 2019-02-14 Integenx Inc. Compositions, procédés, kits et dispositifs d'analyse moléculaire

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EP1167962A1 (fr) * 1999-12-02 2002-01-02 Hymo Corporation Gels de polyacrylamide premoules pour l'electrophorese, procede de production de ces derniers et procede d'electrophorese dans lequel on utilise ces gels
EP1167962A4 (fr) * 1999-12-02 2008-08-06 Hymo Corp Gels de polyacrylamide premoules pour l'electrophorese, procede de production de ces derniers et procede d'electrophorese dans lequel on utilise ces gels
US6939453B2 (en) * 2002-08-14 2005-09-06 Large Scale Proteomics Corporation Electrophoresis process using ionic liquids
EP1533612A1 (fr) * 2004-07-30 2005-05-25 Agilent Technologies, Inc. Matrice de gel pour électrophorese contenant DMSO
US7794576B2 (en) 2004-07-30 2010-09-14 Agilent Technologies, Inc. Protein resolution enhancement by using matrix containing DMSO
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WO2018217816A1 (fr) * 2017-05-22 2018-11-29 Integenx Inc. Compositions, procédés, kits et dispositifs d'analyse moléculaire
WO2018217789A3 (fr) * 2017-05-22 2019-02-14 Integenx Inc. Compositions, procédés, kits et dispositifs d'analyse moléculaire
US20200141901A1 (en) * 2017-05-22 2020-05-07 Life Technologies Corporation Compositions, methods, kits and devices for molecular analysis
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