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WO2008034928A1 - Gel de séparation de protéines - Google Patents

Gel de séparation de protéines Download PDF

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Publication number
WO2008034928A1
WO2008034928A1 PCT/ES2007/000535 ES2007000535W WO2008034928A1 WO 2008034928 A1 WO2008034928 A1 WO 2008034928A1 ES 2007000535 W ES2007000535 W ES 2007000535W WO 2008034928 A1 WO2008034928 A1 WO 2008034928A1
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WO
WIPO (PCT)
Prior art keywords
acrylamide
range
gel
crosslinker
polyacrylamide
Prior art date
Application number
PCT/ES2007/000535
Other languages
English (en)
Spanish (es)
Inventor
José Luís ROSA LÓPEZ
Eduard Casas Terradellas
Francisco Ramón GARCIA GONZALO
Ouadah Hadjebi
Ramón BARTRONS BACH
Francisco Ventura Pujol
Original Assignee
Universidad De Barcelona
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad De Barcelona filed Critical Universidad De Barcelona
Publication of WO2008034928A1 publication Critical patent/WO2008034928A1/fr

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Classifications

    • 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
    • C08F2/00—Processes of polymerisation
    • C08F2/04—Polymerisation in solution
    • C08F2/10—Aqueous solvent
    • 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
    • C08F120/00—Homopolymers 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
    • C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F120/52—Amides or imides
    • C08F120/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F120/56—Acrylamide; Methacrylamide
    • 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
    • C08F265/00—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00
    • C08F265/10—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00 on to polymers of amides or imides
    • 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
    • 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
    • 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
    • G01N27/44782—Apparatus specially adapted therefor of a plurality of samples

Definitions

  • the invention relates to gel electrophoresis and particularly with a polyacrylamide gel for protein separation as well as with a method for obtaining the gel.
  • Gel electrophoresis configures a group of techniques used by scientists to separate molecules based on their physical characteristics such as their size, shape or isoelectric point. Gel electrophoresis is normally used for analytical purposes although it can also be used as a preparatory technique for the partial purification of molecules prior to its use in other methods for its characterization, such as mass spectrometry, PCR (in English, “Polymerase Chain Reaction "), cloning, DNA sequencing or immunoblotting.
  • PAGE polyacrylamide gel electrophoresis
  • This anionic detergent binds along the entire surface of heat-denatured proteins, thereby conferring them a total charge that is proportional to their size (ie, a mass ratio: uniform charge).
  • SDS anionic detergent
  • the migration distance of a protein when an electric current is applied to an SDS-PAGE gel depends solely on the mass of the protein.
  • This fact together with some other refinements such as the use of reducing agents and the use of a concentrating gel has made it possible to separate proteins according to their molecular weight with considerable resolution. To obtain an optimal protein resolution by SDS-PAGE, it is necessary to pour a concentrating gel on the separating gel.
  • the concentrating gel has a lower concentration of acrylamide (larger pore size), a lower pH and a different ionic content. This allows the proteins of each lane to concentrate in a narrow band, before entering the separator gel, producing a gel with narrower and better separated protein bands.
  • SDS-PAGE is a known technique for the separation of small proteins.
  • a gel with a polyacrylamide concentration between 5 and 20% and an acrylamide: bisacrylamide ratio of around 40: 1 is normally used for the analysis of proteins from 5 to 200 kDa (cf. AV Ershov et al., 1996, The Journal of Bioloqical Chemistrv. Vol. 271, pp. 28458-28462).
  • a compound gel with a higher porosity is used. This gel is achieved by increasing the acrylamide: bisacrylamide ratio to 80: 1 for example. A low acrylamide percentage of about 4-5% is also necessary (cf. J. L. Rosa et al., 1996, EMBQ J .. vol. 15, pp. 4262-73).
  • these types of gels are too soft and sticky, which makes them difficult to manipulate in stains or to transfer them into membranes.
  • the present invention relates to a new polyacrylamide gel capable of simultaneously separating high and low molecular weight proteins (i.e., proteins with a molecular weight in the range of 5 kDa to greater than 200 kDa), where this gel comprises a uniform layer formed by different solutions, all polymerized at the same time.
  • high and low molecular weight proteins i.e., proteins with a molecular weight in the range of 5 kDa to greater than 200 kDa
  • the polyacrylamide gel of the invention it is possible to simultaneously analyze giant proteins such as HERC1 (532 kDa) and the heavy chain of the dynein (DYHC, 527 kDa), large proteins such as the heavy chain of the clatrine (CHC, 192 kDa) and small proteins such as ADP ribosylation factor 1 (ARF1, 20 kDa).
  • the polyacrylamide gel of the invention has a good resolution, low manufacturing cost, high reproducibility and saves time compared to the gel systems used in the technique. All these characteristics, together with the fact that a standard apparatus available in any biochemistry laboratory can be used to make the gel of the present invention, makes it an easy and reliable tool.
  • a first aspect of the present invention refers to a polyacrylamide gel comprising: (i) a lower part having a gradient of polyacrylamide concentration within the range of 3% to 20% and an acrylamide: crosslinker ratio selected within from the range of 60: 1 to 20: 1; and (i) a top portion having a polyacrylamide concentration (fixed or gradient) in the range of 3% to 10% and an acrylamide: crosslinker ratio selected within the range of 100: 1 to 70: 1; where both parts form a single layer.
  • single layer refers to a uniform gel with no separation or deformation between the two parts.
  • both parts must be polymerized at the same time.
  • the final gel is a more consistent unit and can be manipulated by taking it through its denser lower region. This is especially important when the gel has to be dyed or transferred to a membrane to perform eg a western blot.
  • a person skilled in the art will easily understand the meaning of the terms “upper part” and "lower part” of a gel.
  • the lower part has a concentration gradient of polyacrylamide within the range of 4% at 18% and an acrylamide: crosslinker ratio selected within the range of 50: 1 to 30: 1.
  • the upper part has a polyacrylamide concentration in the range of 4% to 8% and an acrylamide: crosslinker ratio selected within the range of 90: 1 to 80: 1.
  • the lower part has a gradient of polyacrylamide concentration from 6% to 15% and an acrylamide: crosslinker ratio of 40: 1.
  • the upper part has a polyacrylamide concentration of 4% and an acrylamide: crosslinker ratio of 80: 1.
  • a second aspect of the present invention relates to a process for obtaining the gel of the invention, which comprises the following steps: (a) pouring into a gradient generator solutions comprising acrylamide and crosslinker, suitable for obtaining a concentration gradient of polyacrylamide within the range of 3% to 20% and an acrylamide: crosslinker ratio selected within the range of 60: 1 to 20: 1; (b) adding a second solution comprising acrylamide and crosslinker, suitable for obtaining a polyacrylamide concentration in the range of 3% to 10% and an acrylamide: crosslinker ratio selected within the range of 100: 1 to 70: 1; and (c) allow the solutions of steps (a) and (b) to be polymerized at the same time to form a single layer.
  • the acrylamide that is used to make the solutions of steps (a) and (b) of the process of the invention has to be mixed with a crosslinker in order to form the crosslinked polymer.
  • this polymerization must be done in the presence of a peroxide solution and a reducing agent.
  • the peroxide solution, which is used to form the crosslinked polymer can be, but is not limited to, ammonium peroxodisulfate, and an alkali metal peroxodisulfate.
  • the reducing agents that are used to form the crosslinked polymer may be, but are not limited to, amine compounds, eg N, N, N ', N'-tetramethylethenediamine (TEMED) , N 1 N, - dimethylethylenediamine, 3-dimethylamino-n-propylamine, 3- dimethylaminopropionitrile, Nn-butyldimethylamine and N. N'-dimethylpiperazine.
  • the gradient in step (a) has a polyacrylamide concentration within the range of 4% to 18%, and an acrylamide: crosslinker ratio selected within the range of 50: 1 to 30: 1; and in step (b) the polyacrylamide concentration is in the range of 4% to 8%, and has an acrylamide: crosslinker ratio selected within the range of 90: 1 to 80: 1.
  • step (a) the gradient has a polyacrylamide concentration from 6% to 15%, and an acrylamide: crosslinker ratio of 40: 1; and in step (b) the concentration of polacrylamide is 4% and the acrylamide: crosslinker ratio of 80: 1.
  • crosslinker refers to any compound that has cross-linking functions. Specific examples of compounds may be, but are not limited to, N 1 N'-methylenebisacrylamide (BIS or bisacrylamide), N. N'-propylenenebisacrylamide, diacrylamide dimethyl ether and piperazine diacrylamide. In still a more preferred embodiment of the present invention, the crosslinker is bisacrylamide.
  • a third aspect of the present invention refers to the use of the gel of the invention for the separation of proteins by electrophoresis.
  • the gel of the invention is used to simultaneously separate proteins of very different sizes.
  • the lower part of the gel of the invention is capable of separating proteins whose molecular weights are in the range of 5 kDa to 200 kDa and the upper part of the gel is capable of separating proteins whose molecular weights are greater than 200 kDa, with good resolution.
  • kits for carrying out the process defined above which comprises appropriate amounts of acrylamide, crosslinker and a suitable buffer.
  • a suitable buffer can be Tris / HCI (pH 8.8) + SDS.
  • FIG. 1 A is a schematic representation showing how the gel of the invention is prepared, between two crystals of an apparatus for Western blot ("slab").
  • FIG. 2 shows some applications of the gel.
  • FIG. 2A is a schematic representation of different fusion proteins with GFP, of the giant HERC1 protein and its domains.
  • FIG. 2B and FIG. 2C show plasmids expressing GFP fusion proteins, transfected into HEK-293 cells and analyzed by "immunoblot" with anti-GFP (FIG. 2B) or anti-HERC1 (FIG. 2C) antibodies.
  • FIG. Used 2D sample of HEK-293 cells immunoprecipitated with antibodies against HERC1, preimmune (P) or immune (I).
  • FIG. 1 An example of the gel of the invention is shown in FIG 1.
  • the gel was prepared between two crystals (16 cm x 18 cm) of a standard Western blot apparatus (cf. FWStudier, 2000 TIBS. Vol. 35, pp. 588-590),
  • a standard Western blot apparatus cf. FWStudier, 2000 TIBS. Vol. 35, pp. 588-590
  • three solutions of acrylamide with bisacrylamide were prepared. These solutions had the following characteristics:
  • CMV clathrin-coated vesicles
  • the molecular weight markers are shown in the figures (Myosin: 195 kDa; ⁇ -galactosidase: 116 kDa; Bovine serum albumin: 95 kDa;
  • Ovalbumin 51 kDa; Carbonic anhydrase: 37 kDa; Trypsin inhibitor of soybean seed: 29 kDa; Lysozyme: 20 kDa; Aprotinin: 7 kDa) (BioRad laboratories, Hercules, CA, USA).
  • Example 1 were visualized by staining with Coomassie Brilliant Blue and then were identified by mass spectrometry (MS) and mmunoblot with specific antibodies.
  • MS mass spectrometry
  • the gel was stained with Coomassie Brilliant Blue, whereby the molecular weight markers showed the good resolution of this system (FIG. 1 B).
  • the staining shows a wide range of proteins with very different molecular weights.
  • the subsequent identification by proteomics confirmed the separation in the same gel of giant proteins, such as the heavy chain of the dynein (DYHC, 527 kDa), large proteins such as the heavy chain of the clatrine (CHC, 192 kDa), medium proteins such as Ia chaperone Hsc70 (72 kDa) or synaptotagmine I (Syt I, 65 kDa), and small proteins such as ADP ribosylation factor 1 (ARF1, 20 kDa).
  • giant proteins such as the heavy chain of the dynein (DYHC, 527 kDa), large proteins such as the heavy chain of the clatrine (CHC, 192 kDa), medium proteins such as Ia chaperone Hsc70 (72 k
  • PVDF Polyvinylidene difluoride
  • Plasmids expressing the indicated fusion proteins were transfected into HEK-293 cells (FIG. 2B, 2C). 48 hours later, used of these cells were analyzed with the gel and transferred to a PVDF membrane as previously indicated. GFP fusion proteins were identified by incubation with anti-GFP monoclonal antibodies. (Roche). As shown in FIG. 2B, all GFP fusion proteins were detected in the same membrane, observing as expected a decrease in the degree of expression of fusion proteins directly proportional to their sizes. In this way, smaller fusion proteins such as PFG40 were detected more quickly than larger proteins such as giant protein pFG43, which could be clearly detected with anti HERCI antibodies (FIG. 2C).
  • HERC1 By way of another application of electrophoresis of SDS-PAGE proteins, immunoprecipitation of HERC1 was performed with specific anti-HERC1 antibodies in used HEK-293 cells.
  • HERC1 and its associated proteins CHC: clatrine heavy chain; Hsp 70; CLC: Clatrine light chain
  • CHC clatrine heavy chain
  • Hsp 70 Hsp 70
  • CLC Clatrine light chain

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Electrochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention se rapporte à un gel de polyacrylamide qui comprend : (i) une partie inférieure présentant un gradient de concentration de polyacrylamide compris entre 3 et 20 % et un rapport acrylamide:entrecroiseur sélectionné dans l'intervalle compris entre 60:1 et 20:1; et (ii) une partie supérieure présentant une concentration de polyacrylamide compris entre 3 et 10 % et un rapport acrylamide:entrecroiseur sélectionné dans l'intervalle de 100:1 à 70:1, où les deux parties forment une couche unique. L'invention se rapporte également au procédé et au kit d'obtention de ce gel de polyacrylamide utilisé pour séparer simultanément par électrophorèse des protéines à poids moléculaire élevé et faible.
PCT/ES2007/000535 2006-09-22 2007-09-20 Gel de séparation de protéines WO2008034928A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP200602462 2006-09-22
ES200602462A ES2294949B1 (es) 2006-09-22 2006-09-22 Gel para la separacion de proteinas.

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WO2008034928A1 true WO2008034928A1 (fr) 2008-03-27

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103013982A (zh) * 2012-12-28 2013-04-03 上海启动元生物科技有限公司 一种快速灌制聚丙烯酰胺凝胶的试剂盒及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996032628A2 (fr) * 1995-04-04 1996-10-17 Amresco, Inc. Gels polyacrylamide reticules presentant des rapports monomere/agent de reticulation eleves
US5925229A (en) * 1996-05-03 1999-07-20 The Regents Of The University Of California Low density lipoprotein fraction assay for cardiac disease risk
US20050059761A1 (en) * 2002-02-27 2005-03-17 Bio-Rad Laboratories, Inc., A Corporation Of The State Of Delaware Preparation of defect-free polyacrylamide electrophoresis gels in plastic cassettes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996032628A2 (fr) * 1995-04-04 1996-10-17 Amresco, Inc. Gels polyacrylamide reticules presentant des rapports monomere/agent de reticulation eleves
US5925229A (en) * 1996-05-03 1999-07-20 The Regents Of The University Of California Low density lipoprotein fraction assay for cardiac disease risk
US20050059761A1 (en) * 2002-02-27 2005-03-17 Bio-Rad Laboratories, Inc., A Corporation Of The State Of Delaware Preparation of defect-free polyacrylamide electrophoresis gels in plastic cassettes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CAMPBELL W.P. ET AL.: "Electrophoresis of small proteins in highly concentrated and crosslinked polyacrylamide gradient gels", ANAL. BIOCHEM., vol. 129, 1983, pages 31 - 36, XP024825322, DOI: doi:10.1016/0003-2697(83)90047-7 *
CASAS-TERRADELLAS E. ET AL.: "Simultaneous electrophoretic analysis of proteins of very high and low molecular weights using low-percentage acrylamide gel and a gradient SDS-PAGE gel", ELECTROPHORESIS, vol. 27, no. 20, 20 October 2006 (2006-10-20), pages 3935 - 3938 *
KHALKHALI-ELLIS Z.: "An improved SDS-polyacrylamide gel electrophoresis for resolution of peptides in the range of 3.5-200 KDa", PREP. BIOCHEM., vol. 25, no. 1&2, 1995, pages 1 - 9 *
MARGOLIS J. ET AL.: "Improvement of pore gradient electrophoresis by increasing the degree of cross-linking at high acrylamide concentrations", J. CHROMATOGR., vol. 106, 1975, pages 204 - 209, XP026475087, DOI: doi:10.1016/S0021-9673(01)81066-9 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103013982A (zh) * 2012-12-28 2013-04-03 上海启动元生物科技有限公司 一种快速灌制聚丙烯酰胺凝胶的试剂盒及其应用

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ES2294949B1 (es) 2009-04-01
ES2294949A1 (es) 2008-04-01

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