Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
  • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
  • C08F220/52—Amides or imides
  • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
  • C08F220/56—Acrylamide; Methacrylamide
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
  • C08K5/00—Use of organic ingredients
  • C08K5/16—Nitrogen-containing compounds
  • C08K5/17—Amines; Quaternary ammonium compounds
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S524/00—Synthetic resins or natural rubbers -- part of the class 520 series
  • Y10S524/916—Hydrogel compositions
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S526/00—Synthetic resins or natural rubbers -- part of the class 520 series
  • Y10S526/934—Electrodeposit, e.g. electrophoretic, xerographic

Definitions

• This invention relates to polyacrylamide gels as used in slab gel electrophoresis.
• Slab gels are particularly useful for electrophoresis in view of their ability to accommodate multiple sample analyses and the ease with which the electropherograms can be observed and read by simply identifying the locations of the various bands on the gels that correspond to the individual components.
• Polyacrylamide is a gel material that is widely used in slab gels.
• Slab gels are frequently supplied in pre-cast form, retained between two flat transparent plates in a cassette.
• the plates are typically made of plastic, and a difficulty that has been observed is an apparent distortion of the solute bands due to irregularities in the gel pore size near the interface between the gel and the plastic. These irregularities are detrimental to the electrophoretic analysis since the pore size affects the migration of the solute bands, and pore size variations cause distortions of the bands.
• Polyacrylamide gels are formed from acrylamide monomers and bis-acrylamide crosslinkers by free radical reactions, and molecular oxygen is known to inhibit free radical formation and thereby to limit the growth of the polyacrylamide chain. The problem is particularly acute with polyacrylamide gels formed in plastic enclosures.
• the present invention resides in the discovery that the interface irregularities of polyacrylamide gels that are precast in plastic gel cassettes can be reduced or eliminated by the inclusion of an oxygen scavenger in the gel-forming solution from which the gel is cast.
• the monomer mixture in the solution is polymerized with the scavenger present in the solution, and the result is a pre-cast gel with a substantially uniform pore size throughout.
• Band resolution in the cassette is then comparable to the band resolution that can be obtained with polyacrylamide gels in glass enclosures.
• Oxygen scavengers that can be used in the practice of this invention include many of the materials that have been used or disclosed for use as oxygen scavengers in such applications as boiler operations where they are included for purposes of reducing corrosion. Examples of these materials are sodium sulfite, sodium bisulfite, sodium thiosulfate, sodium lignosulfate, ammonium bisulfite, hydroquinone, diethylhydroxyethanol, diethylhydroxylamine, methylethylketoxime, ascorbic acid, erythorbic acid, and sodium erythorbate.
• Oxygen scavengers of particular interest in the present application are sodium sulfite, sodium bisulfite, sodium thiosulfate, sodium lignosulfate, and ammonium bisulfite, and of these, sodium sulfite and sodium bisulfite are the most preferred.
• the gel-forming solution is an aqueous solution of a monomer mixture that is polymerizable, generally by a free-radical reaction, to form polyacrylamide.
• Monomer mixtures that have been used or are disclosed in the literature for use in forming polyacrylamide gels can be used in the practice of this invention.
• the monomer mixture typically includes acrylamide, a crosslinking agent, and a free radical initiator.
• Preferred crosslinking agents are bisacrylamides, and a particularly convenient crosslinking agent is N,N'-methylene-bisacrylamide.
• the gel-forming solution will also typically include a free radical initiator system. The most common system used is N,N,N',N'-tetramethylenediamine (TEMED) in combination with ammonium persulfate. Other systems will be apparent to those skilled in the art.
• TEMED N,N,N',N'-tetramethylenediamine
• polyacrylamide gels are characterized by the parameters T and C, which are expressed as percents and defined as follows (in which "bis” denotes the bisacrylamide crosslinker): ⁇ _ (combined weight of acrylamide and bis in grams)
• T and C can vary in the present invention as they do in the use of polyacrylamide gels in general.
• a preferred range of T values is from about 5% to about 30%, and most preferably from about 10% to about 20%.
• a preferred range of C values of from about 1% to about 10% (corresponding to a range of weight ratio of acrylamide to bisacrylamide of from about 10:1 to about 100:1), and most preferably from about 2% to about 4% (corresponding to a range of weight ratio of acrylamide to bisacrylamide of from about 25:1 to about 50:1).
• the plastic materials used to form the support plates of the cassettes that will benefit from this invention include a wide variety of plastics.
• the plastics are generally injection moldable plastics, and the selection is limited only by the need for the plastic to be inert to the gel-forming solution, the gel itself, the solutes (typically proteins) in the samples to be analyzed in the cassette, the buffering agents, and any other components that are typically present in the samples.
• these plastics are polycarbonate, polystyrene, acrylic polymers, styrene-acrylonitrile copolymer (SAN, NAS), BAREX® acrylonitrile polymers (Barex Resins, Naperville, Illinois, USA), polyethylene terephthalate (PET), polyethylene terephthalate glycolate (PETG), and poly(ethylene naphthalenedicarboxylate) (PEN).
• the amount of oxygen scavenger included in the gel-forming solution is not critical to the invention and can be varied over a wide range. Certain plastics will require a greater amount of oxygen scavenger than others since the amount of oxygen retained in the plastic varies among different plastics and the manner in which they are formed.
• the optimal amount of oxygen scavenger may also vary with the choice of scavenger. In general, however, best results will be obtained with a concentration of oxygen scavenger that is within the range of from about 1 mM to about 30 mM, and preferably from about 3 mM to about 15 mM, in the aqueous gel-forming solution.
• the amount of oxygen scavenger used may also affect the optimal amounts of the other components.
• oxygen scavengers display catalytic activity toward the free radical reaction, and a lower concentration of free radical initiator can then be used when such scavengers are present.
• concentrations of TEMED and ammonium persulfate can be lower than would otherwise be used.
• the standard protein mixture was myosin (200 kD), ⁇ -galactosidase (116 kD), phosphorylase B (97.4 kD), bovine serum albumin (66.2 kD), ovalbumin (45 kD), carbonic anhydrase (31 kD), trypsin inhibitor (21.5 kD), and lysozyme (14.5 kD).
• SDS-PAGE electrophoresis

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• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Molecular Biology (AREA)
• General Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• Electrochemistry (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Medicinal Chemistry (AREA)
• Polymers & Plastics (AREA)
• Dispersion Chemistry (AREA)
• Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
• Medicinal Preparation (AREA)
• Polymerisation Methods In General (AREA)
• Solid-Sorbent Or Filter-Aiding Compositions (AREA)
• Compositions Of Macromolecular Compounds (AREA)
• Separation Using Semi-Permeable Membranes (AREA)

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• 2002
  • 2002-02-27 US US10/087,140 patent/US6846881B2/en not_active Expired - Lifetime
• 2003
  • 2003-02-26 WO PCT/US2003/006207 patent/WO2003072616A1/en not_active Ceased
  • 2003-02-26 EP EP03709407A patent/EP1478670B1/en not_active Expired - Lifetime
  • 2003-02-26 JP JP2003571320A patent/JP4287284B2/ja not_active Expired - Fee Related
  • 2003-02-26 AU AU2003212467A patent/AU2003212467C1/en not_active Ceased
  • 2003-02-26 DE DE60322027T patent/DE60322027D1/de not_active Expired - Lifetime
  • 2003-02-26 CA CA002476768A patent/CA2476768C/en not_active Expired - Fee Related
• 2004
  • 2004-10-25 US US10/973,541 patent/US20050059761A1/en not_active Abandoned

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