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WO2004020991A1 - Gel d'electrophorese presentant des proprietes de gonflement ameliorees - Google Patents

Gel d'electrophorese presentant des proprietes de gonflement ameliorees Download PDF

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Publication number
WO2004020991A1
WO2004020991A1 PCT/AU2003/001122 AU0301122W WO2004020991A1 WO 2004020991 A1 WO2004020991 A1 WO 2004020991A1 AU 0301122 W AU0301122 W AU 0301122W WO 2004020991 A1 WO2004020991 A1 WO 2004020991A1
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Prior art keywords
gel
ipg
gradient
immobilised
organic acid
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PCT/AU2003/001122
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English (en)
Inventor
Benjamin Ross Herbert
Maurizio Bruschi
Luca Musante
Giovanni Candiano
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Proteome Systems Intellectual Property Pty Ltd
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Publication date
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Priority to AU2003257238A priority Critical patent/AU2003257238A1/en
Publication of WO2004020991A1 publication Critical patent/WO2004020991A1/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
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing
    • 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 an improved gel for electrophoresis and to a method of preparing an improved gel.
  • the present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, polymer science, protein biochemistry, and protein separation as described in, for example,
  • one-dimensional and two-dimensional gel electrophoresis have become standard tools for separating and visualising macromolecules.
  • the highest resolution method for separating macromolecules is two- dimensional electrophoresis.
  • Such two-dimensional electrophoresis usually involves sequential separations in a first dimension by isoelectric focusing and in a second dimension by SDS gel electrophoresis.
  • the standard method of performing the first dimension is isoelectric focusing using an immobilised pH gradient (IPG).
  • immobilised pH gradients are polyacrylamide gels in which buffering groups responsible for the formation of the pH gradient are acrylamide derivatives co- polymerised into the gel with acrylamide and a cross-linker. These derivatives are called “Immobilines” by the manufacturer, Pharmacia. A gradient of these "Immobiline” groups is cross-linked to polyacrylamide.
  • the polyacrylamide of commercial IPGs are cross-linked by bis-acrylamide or piperazine di-acrylamide (PDA) and often have an acrylamide concentration of 4%T.
  • the polyacrylamide gels are attached to an activated backing sheet, dried and cut into strips, approximately 3mm wide. In use, the dry strips are rehydrated in a protein solution.
  • IPGs immobilised pH gradients
  • Proteomic studies also require gels with varying pore size in order to separate and analyse small and large molecules. While commercial gels having relatively small pore size are readily available, the production of gels having large pore sizes continues to have its setbacks. Previous studies have shown that the pores in the gel, as a function of %C B . S , progressively open up at both very low and very high cross-linker values. The relationship between pore size and %T, by contrast, is quite straightforward and assumes an almost linear decay as the %T is progressively increased. A third way for dramatically enlarging pore sizes would be to produce laterally-aggregated matrices, i.e. polyacrylamides that are forced to produce bundles of fibers, during the gelling process, via addition of preformed polymers in the polymerizing solution.
  • laterally-aggregated matrices i.e. polyacrylamides that are forced to produce bundles of fibers, during the gelling process, via addition of preformed polymers in the polymerizing solution
  • an IPG gel could be produced having large pore size which is easily and uniformly reswollen.
  • the present invention provides an immobilised pH gradient (IPG) gel that is more uniformly reswollen than currently available gels. Furthermore, the improved IPG gel of the present invention can be produced with larger pore sizes than their conventional equivalents. The combined effects of more uniform reswelling and of diluting the gel matrix favour penetration of large macromolecules and allows for better spot resolution and for the display of a substantially higher number of spots on the IPG gel.
  • the present invention provides an immobilised pH gradient (IPG) gel comprising an organic acid or organic base, wherein when hydrated, the IPG gel is substantially uniformly swollen along the pH gradient of the gel.
  • IPG immobilised pH gradient
  • the term "hydrated” includes rehydration of a gel from a partially or fully dehydrated state.
  • the organic acid is a carboxylic acid
  • the present invention provides an immobilised pH gradient (IPG) gel comprising (i) polymerised monomeric units of CH 2 CR ⁇ CONR 2 R 3 and cross-linker and (ii) an organic acid or organic base, wherein Ri, R 2 and R are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different, and wherein when hydrated the IPG gel is substantially uniformly swollen along the pH gradient.
  • the IPG gel of the present invention is capable of being dehydrated.
  • the IPG gel of the present invention is capable of being rehydrated.
  • the invention provides an IPG gel comprising
  • the dehydrated IPG gel is stable and can be stored for a period of time before being rehydrated and used.
  • an organic acid or base is contacted with a polymerised gel.
  • an organic acid or base can be mixed with an IPG gel mixture prior to or during the polymerisation of a gel matrix.
  • the polymerised IPG gel is washed in water to remove unreacted monomers and/or organic acid or organic base.
  • the present invention provides a method of
  • IPG immobilised pH gradient
  • the present invention provides a method of preparing an IPG gel, the method comprising contacting a polymerised gel with an organic acid or organic base, wherein when hydrated the IPG gel is substantially uniformly swollen along the pH gradient.
  • the present invention provides a method of preparing a dehydrated IPG gel, the method comprising contacting a polymerised IPG gel with an organic acid or organic base, and then dehydrating the polymerised gel.
  • the present invention provides a method of preparing an IPG gel, the method comprising: polymerising a mixture of at least one monomer of formula CH 2 CR ⁇ CONR 2 R 3 and at least one cross-linker to form a gel matrix, wherein Ri, R 2 and R are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different; and contacting the gel matrix with an organic acid or organic base, such that an amount of the organic acid or organic base is adsorbed in the gel matrix.
  • the method further comprises dehydrating the gel matrix to form a dehydrated IPG gel.
  • the present invention provides a method of preparing an IPG gel, the method comprising:
  • the method further comprises dehydrating the gel matrix after step (b), to form a dehydrated IPG gel.
  • the present invention provides an immobilised pH gradient (IPG) gel obtainable by any of the methods of the present invention, wherein when hydrated the IPG gel is uniformly swollen along the pH gradient.
  • IPG immobilised pH gradient
  • the present invention provides use of an immobilised pH gradient (IPG) gel according to the present invention for separating and/or analysing a mixture of macromolecules.
  • IPG immobilised pH gradient
  • the present invention provides a kit for analysing or separating macromolecules in a mixture, the kit comprising one or more electrophoresis gel plates according to the first aspect of the invention, buffers and optionally instructions for use.
  • Fig. 1 is a photograph of two pH 3-10 IPG gels, the one to the left subjected to the usual washing in plain water, the one to the right, instead, equilibrated for 30 min in 100 mM citric acid, followed by two rinses in distilled water.
  • Fig. 2 is a photograph of two 2-D maps of human platelets run in soft (T% ⁇ 4) IPG strips, rinsed in plain distilled water (left panel) or in 100 mM citric acid (right panel).
  • Fig. 3 is a photograph of 2-D maps of human platelets run in commercial IPG strips (left panel) or in soft IPG gel (T% ⁇ 4) strips, rinsed in 100 mM citric acid (right panel).
  • Fig. 4 is a graph of IPG reswelling versus pH gradient, wherein "dry" commercial IPG gels were measured (cast volume), and then rehydrated and measured again (maximum rehydration volume) .
  • the present invention provides an improved IPG gel comprising an organic acid or organic base, wherein when hydrated the IPG gel is substantially uniformly swollen along the pH gradient.
  • the organic acid is a carboxylic acid.
  • the carboxylic acid is a polycarboxylic acid.
  • the polycarboxylic acid may be of the formula R(COOH) n , where R is branched or unbranched optionally substituted alkyl, branched or unbranched optionally substituted alkenyl group, or branched or unbranched optionally substituted alkynyl group and n is an integer from 1 to 4.
  • R is C 2 to C 5 alkyl.
  • a substituent is -OH.
  • a particularly preferred organic acid is one of formula:
  • R 2 are independently selected from Ci to C 3 alkyl; R 3 is selected from OH, or R 4 OH, where R 4 is Ci to C 3 alkyl; and X is absent or is lower alkyl.
  • a particularly preferred organic acid is citric acid.
  • the organic base comprises an amine group. Alternate basic groups are not excluded.
  • alkyl group refers to a saturated aliphatic hydrocarbon radical including straight-chain, branched chain, cyclic groups, and combinations thereof.
  • Typical alkyl groups and substituted alkyl groups include but are not limited to methyl, ethyl, 1-propyl, isopropyl, 1 -butyl, 2-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl and octyl.
  • alkenyl group refers to an unsaturated aliphatic hydrocarbon radical including straight, branched chain, cyclic groups, and combinations thereof, having at least one double bond, of either E or Z stereochemistry where applicable.
  • alkenyl include but are not limited to ethenyl, 1- propenyl, 2-propenyl, 2-methyl-2-propenyl, 1-butenyl, 1,3-butadienyl, hexenyl, pentenyl, heptenyl and octenyl.
  • alkynyl group refers to an unsaturated aliphatic hydrocarbon group including straight, branched chain, cyclic groups and combinations thereof, having at least one triple bond.
  • alkynyl groups include but are not limited to include ethynyl, 1-propynyl, 1-, and 2-butynyl,and l-methyl-2-butynyl.
  • substituted refers to replacing a group with another group, for example, a carbon group can be replaced with a heteroatom or halide or -OH.
  • heteroatom refers to any atom other than carbon or hydrogen and preferably means oxygen, nitrogen or sulphur.
  • halide atom refers to fluorine, chlorine, bromine or iodine.
  • amine group refers to the group -NR 5 R 6 wherein R 5 and R 6 are individually selected from the group including but not limited to H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, alkynyl and substituted or unsubstituted aryl groups.
  • the IPG gel according to the present invention may comprises trace amounts of organic acid or organic base.
  • the IPG gel comprises from about 1 ⁇ M to about 500mM, more preferably about 1 ⁇ M - lOOmM .
  • the concentration of organic acid or organic base in the IPG gel is expected to vary over the length of the IPG gel.
  • a polymerised IPG gel is contacted with an organic acid or organic base in an effective concentration and for a length of time such that an effective amount of organic acid or organic base is adsorbed by the IPG gel.
  • contacting includes for example, washing, spraying, bathing, and immersing.
  • the concentration of organic acid or organic base contacted with the IPG gel may be from micromolar to molar concentrations.
  • the amount of time that the IPG gel is in contact with the organic acid or organic base is variable, and can be anything from a few seconds to hours. It is understood, therefore that the gel is contacted with an amount of organic acid or organic base for an effective amount of time that is sufficient for an amount of organic acid or organic base to be adsorbed by the gel. Excess organic acid or organic base can be removed, for example, by washing the gel in water.
  • the gel is contacted with about lOmM-lM organic acid or organic base.
  • the gel is contacted with about 50mM-500mM organic acid or organic base, more preferably about 50mM-300mM organic acid or organic base, more preferably about 75mM-200mM organic acid or organic base, most preferably about 50mM-100mM organic acid or organic base, more preferably about lOOmM.
  • the gel is contacted with an amount of organic acid or organic base for about 5 seconds - 5 hours. More preferably, the gel is contacted with an amount of organic acid or organic base for about 1 minute - 3 hours, more preferably about 10 minutes - 1 hour, more preferably about 20 minutes - 45 minutes, more preferably about 30 minutes.
  • the present invention provides a method of preparing an IPG gel, the method comprising including an organic acid or organic base in a mixture of monomers of CH 2 CR ⁇ CONR 2 R 3 and cross-linker.
  • the effective concentration of organic acid or organic base included in the mixture will vary from micromolar to molar concentrations, such as for example, about 1 ⁇ M to about 500mM organic acid or organic base.
  • the gel can be washed in, for example water, to remove any unreacted monomers, initiators and organic acids or organic bases.
  • the IPG gel is hydrated, rehydrated or dehydrated.
  • hydrated and “hydration” refer to the process whereby ions (and other species) in aqueous solutions are solvated by water. According to the present invention it is understood that when used for electrophoresis, IPG gels are used in a hydrated state. According to the invention, when hydrated the IPG gel is substantially uniformly swollen along the pH gradient of the IPG gel. As used herein “substantially uniformly” refers to the gel being substantially evenly swollen, and preferably more evenly swollen compared to currently available IPG gels.
  • the property of being uniform applies to the thickness of the gel. Accordingly, preferably the thickness of a rehydrated IPG gel of the present invention is substantially uniform along the pH gradient of the gel.
  • the pH gradient of an IPG gel provides an IPG gel having a more alkaline end and a more acidic end.
  • addition of an organic acid enhances the reswelling properties of the alkaline end of an IPG gel.
  • the alkaline end of the hydrated IPG gel can be thicker than the acidic end after organic acid treatment.
  • the alkaline end is overswollen compared to the acidic end, wherein the rippled effect is evidence of overswelling.
  • the invention comprises an improved hydrated IPG gel comprising an organic acid or organic base, the gel having an alkaline end and an acidic end, wherein the alkaline end has the same or greater thickness as the acidic end.
  • the alkaline end and acidic end have substantially the same thickness.
  • dehydrated refers to gels having the water present during the polymerisation and washing substantially removed.
  • a gel can shrink to a thickness less than or equal to 10% of their, cast thickness.
  • a dehydrated IPG can reswell with a volume equal to it's cast volume.
  • an 11cm IPG 110mm x 3.3mm x 0.5mm
  • the immobilised pH gradient (IPG) gel comprises polymerised monomeric units of CH 2 CR 1 CONR2R3 wherein Ri, R 2 and R 3 are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different.
  • the monomeric units are selected from any of acrylamide, Dimethylacrylamide (DMA) and N-Acryloyl amino propanol (AAP).
  • DMA Dimethylacrylamide
  • AAP N-Acryloyl amino propanol
  • Other monomers according to the formula are not excluded.
  • Mixtures of monomeric units are encompassed by the present invention.
  • the invention is intended to include mixtures of monomers, for example, as described in PCT/AU02/00666 (incorporated herein by reference).
  • PCT/AU02/00666 describes a hybrid IPG gel comprising a mixture of hydrophilic and hydrophobic monomers to produce improved IPG gels.
  • the IPG gels described in PCT/AU02/00666 are more hydrophobic than commercially available gels, preferably amphiphilic and have improved stability.
  • the described gels can preferably be used with strong hydrophobic extraction solutions such as sulfolane, which provides the ability to extract a wider range of proteins and other macromolecules from a sample.
  • strong hydrophobic extraction solutions such as sulfolane
  • Methods for making or casting gels are well known in the art.
  • acrylamide gel matrix compositions are described as %T/%C, wherein T is the total acrylamide and C is the amount of crosslinking agent.
  • the gel matrix comprises about 2.5-10.0% total acrylamide concentration at a cross-link density of 2-15%.
  • the gel matrix comprises about 2.5-8% total acrylamide concentration.
  • the gel matrix comprises about 2.5-7% total acrylamide concentration, more preferably about 2.5-6% total acrylamide concentration, more preferably about 3-5% total acrylamide concentration.
  • the gel matrix comprises about 4% total acrylamide concentration.
  • the immobilised pH gradient (IPG) gel comprises a cross- linker.
  • cross linkers are known by the skilled addressee, including for example, cross-linkers used in commercial IPGs such as bis-acrylamide, diacroyl piperazine, DATD, N,N'-diallyl-tartardiamide or piperazine di-acrylamide (PDA).
  • BAG bis-acryloyl cystamine
  • Other cross-linkers are not excluded.
  • the crosslinker is 1,4-Bis (acryloyl)piperazine.
  • the cross-link density is about 2-15%. More preferably, the crosslink density is about 3-12%, more preferably about 5-10%,. Most preferably the cross- link density is about 6%.
  • the gel matrix comprises 3.75%T/6%C polyacrylamide solution. That is, the gel matrix comprises 3.75% total acrylamide of which 6% is from cross-linking 1,4-Bis (acryloyl)piperazine.
  • the invention is intended to include IPG gels comprising any suitable pH gradient.
  • the immobilised pH gradient is in the range of about pH 2 to 12.
  • the immobilised pH gradient can be selected from a range of pH gradients including, for example about pH 2-10, about pH 3-10, about pH
  • the immobilised pH gradient gel is an IPG gel strip or an IPG gel strip
  • a gel slab is a whole sheet of gel as polymerised.
  • a strip is typically a narrow (between 3 and 10mm wide) piece of a slab cut out and used for 2-D gels.
  • a slab is anything wider than what would be typically used for 2-D gels.
  • commercial IPGs are 3.3mm wide.
  • an IPG gel strip is attached to a backing sheet.
  • the backing sheet is a plastic backing sheet.
  • the plastic is treated plastic.
  • the treatment causes the polymerising acrylamide to crosslink to the plastic and thus helps to further stabilise the gel.
  • IPG gels according to the present invention are tested for stability according to standard methods as described in
  • IPG gels according to the invention are tested for chemical stability to acidic or alkaline conditions.
  • the present invention provides a kit for analysing or separating macromolecules in a mixture, the kit comprising one or more IPG gels according to the first aspect of the invention, buffers and optionally instructions for use.
  • the macromolecule is a proteinaceous macromolecule, preferably in a mixture of proteins.
  • the kit further comprises any one or more of the following: urea, thiourea, CHAPS, carrier ampholytes and an electrophoresis apparatus.
  • CHAPS iodoacetamide
  • TBP tributylphosphine
  • SDS sodium dodecyl sulfate
  • Tris(hydroxymethyl)aminomethane and DL-dithiothreitol (DTT) were from Sigma, St. Louis, MO, USA. All the Ampholines, bromophenol blue and agarose were from Pharmacia-LKB (Uppsala, Sweden).
  • Acrylamide, N,N'-methylenebisacrylamide and N,N,N'N'-tetramethylethylenediamine (TEMED) were from Bio-Rad Labs (Richmond, CA).
  • Trichloro acetic acid (TCA), Acetone, Chloroform, methanol and citric acid were from Merck (Darmstadt, Germany).
  • the support is gel bond pag-film
  • the gel was dried at room temperature with a fan or ventilator. Once the gel was dry, it was covered with mylar film and stored at - 20 D C
  • Platelet preparation Fresh whole blood was collected from normal healthy volunteers. Each blood sample was processed individually and was mixed with a sodium citrate stock solution to a final concentration of 10% v/v of the anticoagulant. Platelets isolation was ca ⁇ ied out as previously described in Gibbins, J., Asselin; J., Famdale, R require Barnes, M., Law, C. L.,Watson, S. P. J Biol Chem 1996, 271, 18095-18099.
  • ACD acid citrate dextrose
  • the platelets were washed in Tyrode— HEPES (134 mM NaCl, 0.34 mM Na 2 HPO 4 , 2.9 mM KCl, 12 mM NaHCO 3 , 20 mM HEPES, 5 mM glucose, 1 mM MgCl 2 pH 7.3 and EGTA 1 mM) containing ACD (7%, v/v).
  • the platelet pellet was then resuspended in Tyrodes- HEPES, to a concentration of 2 ⁇ l0 s /ml or 1 xlO ⁇ /ml and following addition of 20 1 of a protease inhibitor cocktail with immediately frozen in liquid nitrogen prior to storage at -80°C. Pellets of frozen platelets was reduced, alJ ⁇ lated and delipidated, prior isoelectric focusing .
  • the protein platelets of the stock preparation (5 ⁇ g/ ⁇ l) were added to a 10% SDS, 3% DTE, 40 M Tris and 0.1 mM EDTA solution and treated for 5 min at 100 Q C in a method as escribed .Herbert B. et al., Electrophoresis 2001, 22, 204-57. 5 Preparation of dilipidated platelets for 2-D PAGE
  • the sample was dilipidated with a solution consisting of tri-n-burylphosphate:acetone:methanol (1:12:1) and cooled in ice. Fourteen (14) mL of this mixture were added to the SDS-solubilized platelets to a final acetone concentration of 80% and incubated at 4°C for 90 min. The precipitate was pelleted by centrifugation at 2800 g for 20 min at 4°C. After washing the pellet with the same delipidizing solution, it was centrifuged again and then air-dried. The pellet was finally dissolved in the focusing solution, i.e. 8 M urea, 1 M thiourea, 4% w/v CHAPS, 65 mM
  • Proteome IPG strips having the same size and IPG composition as the commercial
  • IPG strips but made with variable matrix content, ranging from as low as 3%T up to
  • IPG strips All samples were routinely loaded, onto caps at the cathodic end of the rehydrated IPG strips, and covered with low-viscosity paraffin oil. IEF was run at 18°C. The voltage was progressively increased from 300 to 3000 N during the first 3 hrs, followed by 5000 N for a total of 100 kV. Before the 2 nd dimension run, the IPG strips were equilibrated for 15 min with a solution of 40 mM Tris-acetate buffer, pH 6.8 containing 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and 2.6% (w/v) DTE.
  • proteins were separated based on their size in 8-18%T gradient polyacrylamide gels having the following dimensions: 180 x 160 x l.5 mm.
  • the electrophoretic equipment included a Protean II Multi-Cell vertical chamber and a power supply 3000x1 (Bio-Rad, Hercules, CA, USA). No stacking gel was employed.
  • the IPG strips were embedded with 0.5 % (w/v) melted agarose prior to the run on the SDS-PAGE slabs.
  • the agarose contained 0.001 % (w/v) bromophenol blue as a tracking dye.
  • the gels were run at 45 mA/gel constant current and maintained at a temperature between 8 and 12°C.
  • the proteins were visualized by a double staining procedure: first the methyl-trichloroacetate negative staining of Candiano, G., Porotto, M., Lanciotti, M., Ghiggeri, G.M., Anal. Biochem. 1996, 243, 245-250, followed by the silver staining of Oakley, B.R., Kirsch, D.R., Morris, N.R., Anal. Biochem. 1980, 105, 361-366.
  • Results Fig. 1 compares two pH 3-10 IPG gels, the one to the left subjected to the usual washings in plain water, the one to the right, instead, equilibrated for 30 min in 100 mM citric acid, followed by two rinses in distilled water. It can be noticed that the latter one, in the alkaline region, shows the typical ripples of a slight overswelling, as though it were more charged than the acidic counter-part.
  • the IPG strips washed in citric acid exhibited a much better tendency to reswell and become impregnated with sample solution.
  • Fig. 2 displays 2-D maps of purified human platelets.
  • Two major improvements can be appreciated in the gels which had undergone citric acid washing: the alkaline gel region in richer in spots; overall, more spots appears throughout the entire gradient and high M r macromolecules (>200 kDa) are revealed as much more intense spots.

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Abstract

La présente invention concerne un gel à gradient de pH immobilisé (IPG) qui se regonfle de manière plus uniforme que les gels actuellement disponibles. En outre, le gel IPG amélioré de la présente invention peut être produit avec des dimensions des pores plus importantes par comparaison avec les équivalents classiques. Par conséquent, de manière générale, la présente invention concerne un gel à gradient de pH immobilisé (IPG) comprenant un acide organique ou une base organique. Lorsqu'il est hydraté, ce gel IPG se gonfle de manière sensiblement uniforme le long du gradient de pH.
PCT/AU2003/001122 2002-09-02 2003-09-01 Gel d'electrophorese presentant des proprietes de gonflement ameliorees WO2004020991A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2267502A (en) * 1992-05-28 1993-12-08 Aligena Ag Immobilized buffered gels and membranes of hydroxy groups containing polymers
WO1997045445A1 (fr) * 1996-05-30 1997-12-04 Proteome Sciences Plc Marqueur proteique pour cancer de l'oesophage
WO1998035985A1 (fr) * 1997-02-12 1998-08-20 The Regents Of The University Of Michigan Proteines-marqueurs pour le cancer du poumon et utilisation de ces dernieres
WO2001060841A1 (fr) * 2000-02-18 2001-08-23 Gradipore Limited Gels d'electrophorese ameliores
WO2002056003A2 (fr) * 2001-01-12 2002-07-18 Curagen Corporation Procede et formulations pour la separation de macromolecules biologiques
WO2002056991A2 (fr) * 2001-01-16 2002-07-25 Arqule, Inc. Systemes et procedes de chromatographie electrocinetique
WO2002096931A1 (fr) * 2001-05-25 2002-12-05 Proteome Systems Intellectual Property Pty Ltd Solubilisation accrue de proteines hydrophobes
WO2002103345A1 (fr) * 2001-06-14 2002-12-27 Proteome Systems Intellectual Property Pty Ltd Gel ameliore pour electrophorese et ses utilisations

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2267502A (en) * 1992-05-28 1993-12-08 Aligena Ag Immobilized buffered gels and membranes of hydroxy groups containing polymers
US5599506A (en) * 1992-05-28 1997-02-04 Aligena Ag Immobilized buffered gels and membranes of hydroxy groups containing polymers
WO1997045445A1 (fr) * 1996-05-30 1997-12-04 Proteome Sciences Plc Marqueur proteique pour cancer de l'oesophage
WO1998035985A1 (fr) * 1997-02-12 1998-08-20 The Regents Of The University Of Michigan Proteines-marqueurs pour le cancer du poumon et utilisation de ces dernieres
WO2001060841A1 (fr) * 2000-02-18 2001-08-23 Gradipore Limited Gels d'electrophorese ameliores
WO2002056003A2 (fr) * 2001-01-12 2002-07-18 Curagen Corporation Procede et formulations pour la separation de macromolecules biologiques
WO2002056991A2 (fr) * 2001-01-16 2002-07-25 Arqule, Inc. Systemes et procedes de chromatographie electrocinetique
WO2002096931A1 (fr) * 2001-05-25 2002-12-05 Proteome Systems Intellectual Property Pty Ltd Solubilisation accrue de proteines hydrophobes
WO2002103345A1 (fr) * 2001-06-14 2002-12-27 Proteome Systems Intellectual Property Pty Ltd Gel ameliore pour electrophorese et ses utilisations

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