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WO2001036449A1 - Appareil d'electrophorese a plusieurs compartiments - Google Patents

Appareil d'electrophorese a plusieurs compartiments Download PDF

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Publication number
WO2001036449A1
WO2001036449A1 PCT/AU2000/001391 AU0001391W WO0136449A1 WO 2001036449 A1 WO2001036449 A1 WO 2001036449A1 AU 0001391 W AU0001391 W AU 0001391W WO 0136449 A1 WO0136449 A1 WO 0136449A1
Authority
WO
WIPO (PCT)
Prior art keywords
chambers
chamber
isoelectric
series
magnetic stirrer
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/AU2000/001391
Other languages
English (en)
Inventor
Ben Herbert
Pier Giorgio Righetti
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Proteome Systems Ltd
Original Assignee
Proteome Systems Ltd
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
Priority claimed from AUPQ4058A external-priority patent/AUPQ405899A0/en
Priority claimed from AUPQ8727A external-priority patent/AUPQ872700A0/en
Application filed by Proteome Systems Ltd filed Critical Proteome Systems Ltd
Priority to AU12589/01A priority Critical patent/AU755326B2/en
Priority to JP2001538938A priority patent/JP2003514829A/ja
Priority to EP00974180A priority patent/EP1230258A4/fr
Priority to HK03100703.5A priority patent/HK1049343A1/zh
Publication of WO2001036449A1 publication Critical patent/WO2001036449A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
    • B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—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
    • G01N27/44708—Cooling

Definitions

  • the present invention relates to an apparatus and a method utilising that apparatus for sub-fractionation and subsequent separation of the fractions from highly complex protein/peptide mixtures, such as those found in total cell lysates.
  • body fluids e.g.. plasma, sera, cerebrospinal fluid, urine
  • tissue extracts in general.
  • the present invention seeks to provide a new instrument which is capable of being operated to provide improvements in resolving power and preferably improvements in detection sensitivity.
  • a novel multi-compartment electrolyser design to pre-fractionate complex protein mixtures, for use prior to the implementation of 2-D maps.
  • Such a sub-fractionation processes can effectively remove, via suitable narrow range isoelectric membranes, proteins present in large excess in a cell lysate or in body fluids.
  • the remaining protein mixture devoid of such major components, can be loaded in a narrow pH range 2-D map at much higher levels, thus ensuring a greater sensitivity and detection capability of low-abundance proteins.
  • the apparatus embodying the present invention produces protein fractions which are fully compatible with the subsequent 2-D protocols, since it is based on a focusing technique, which yields samples highly concentrated and devoid of salts and buffers.
  • the process of the present invention may be carried out using a multi- compartment electrolyser. which is characterised in that recirculation of the samples is not required.
  • the present invention provides a process for electrophoretic separation of a mixture of macromolecules into groups comprising the steps of placing a sample mixture of macromolecules in a separation apparatus comprising a series of chambers separated by isoelectric membranes of known pi.
  • the apparatus also having chambers located at each end of the series of chambers containing means for applying an electric field across the series of chambers: and applying an electric field across the chambers to separate the macromolecules on the basis of their isoelectric point characterised in that the sample mixture is agitated within the chambers and is not recirculated
  • Each chamber preferably defines a well or recess adapted to receive a magnetic stirrer bar.
  • Electrodecantation may be prevented by placing the device in a multi- place magnetic stirring platform and mixing the contents of each chamber of the electrolyser with the magnetic stirrer bar.
  • the separator apparatus of the present invention may utilise an associated platform which provides power, cooling and a magnetic stirrer platform.
  • the location of the magnets in the magnet array of the magnetic stirrer platform have to coincide with the location of the stirrer magnets in the electrolyser.
  • Heat dissipation is implemented by the use of embedded Peltier elements in the magnetic stirrer platform which can be regulated to maintain the correct temperature.
  • the platform may also be configured to accommodate a plurality of gel trays for isoelectric focusing in carrier ampholyte pH gradients or immobilised pH gradients.
  • the multi- compartment electrolysers are assembled from a plurality of separate chambers, operating in an electric field, with a set of isoelectric membranes (having pi values increasing monotonically from anode to cathode) able to trap a desired protein population within a given chamber.
  • the pre- fractionation mode using one or more multi-compartment electrolysers.
  • the device can be operated under denaturing conditions (with mixtures of chaotropes.
  • the temperature will be set to about 20°C.
  • the device can be operated under native conditions, in the absence of denaturants. when native proteins are required for further analysis exploiting biological activity and this it typically done at about 4°C.
  • the isoelectric membranes may be made either with standard acrylamide monomers or with stable acrylamide derivatives, such as N- acryloyl amino ethoxy ethanol or N-acryloyl amino propanol. or mixtures thereof.
  • Membrane pH is defined by a suitable choice of buffering and counter-ion species, which are acrylamido-derivatives able to be copolymerised with polyacrylamide, or its derivatives, and possessing good buffering power.
  • the isoelectric membranes may be made macroporous either by high levels of cross linkers, or by lateral aggregation of the acrylamide chains or by a combination of both means.
  • the sub-fractionation of complex protein/samples may be obtained under native conditions in presence of suitable solubilizers compatible with maintenance of protein integrity, such as sugars, non-detergent sulfobetaines. glycerol, ethylene and propylene glycols and mixtures thereof.
  • the sub-fractionation of complex protein/peptide mixtures may be obtained under denaturing conditions in presence of appropriate solubilizing cocktails, such as organic solvents, chaotropes. neutral and zwitterionic surfactants and amido-sulfobetaines and, when required, in presence of suitable disulfide bridge reducing and/or alkylating agents.
  • solubilizing cocktails such as organic solvents, chaotropes.
  • neutral and zwitterionic surfactants and amido-sulfobetaines and, when required, in presence of suitable disulfide bridge reducing and/or alkylating agents.
  • the preferred embodiment for sample application may be by pulse- loading in a single chamber of the electrolyser, said chamber being preferably in the neutral or basic region of the pH scale.
  • the sub-fractionation process may be performed in a cascade fashion, such that a wider pH fraction obtained in a first run in the electrolyser. and may subsequently be further fractionated in a very narrow pH window, as required by the sample complexity.
  • the device described in the present invention also comprises a second operational mode for simultaneous separation of certain protein fractions, using isoelectric focusing in carrier ampholyte pH gradients or immobilised pH gradients.
  • a second operational mode for simultaneous separation of certain protein fractions, using isoelectric focusing in carrier ampholyte pH gradients or immobilised pH gradients.
  • Figure 1 is a schematic exploded view of a multi-compartment electrolyser apparatus embodying the present invention
  • Figure la illustrates a suspended magnetic stirrer element
  • Figure 2 is a schematic view of two multi-compartment electrolysers set up accommodated on an electrophoresis platform containing a power supply, Peltier cooling and a multi-place magnetic stirrer;
  • Figure 3 is a schematic view of two trays for isoelectric focusing accommodated on an electrophoresis platform containing a power supply; Peltier cooling and a multi-place magnetic stirrer:
  • Figure 4 is a silver stained 2-D map of a pi 4-5 fraction of E. coli. as purified in the multi-compartment electrolyser of Fig. 1, run in a 7cm pH 3- 10 IPG strip in the first dimension:
  • Figures 5a and 5b are 2-D maps of a plasma sample in a narrow pH 3-6, first dimension Immobiline gel. with Figure 5a showing unfractionated plasma and Figure 5b showing pre-fractionated plasma.
  • Figure 1 shows a disassembled separation apparatus in the from of a multi-compartment electrolyser apparatus 10.
  • the apparatus includes five chamber blocks, defining three inner fractionation chambers 12 and two. outer, electrode chambers 14.
  • Each chamber block is generally square in transverse cross-section.
  • a cylindrical through bore 16 extends through the centre of each of the fractionation chamber blocks and part way into the outer electrode chambers 14.
  • Four narrower through bores 18 located at the corners of the square cross-section of the blocks extend through all five chambers.
  • the bores 18 receive threaded tie rods (not shown) which can be used to align the bores together and join the chambers together.
  • Each chamber also includes a sample inlet 26 in the upper face of the chamber block which extends from the upper face of the block down to the bore 18.
  • Each chamber 12 defines a shallow well or recess 20 at the base of the bore which is typically co-axial with the sample inlet. In use. the well receives a magnetic stirrer. not illustrated.
  • each chamber block 12 defines an annular spigot 22 extending around the bore 16.
  • the opposite side defines a corresponding recess 24.
  • the open side of one electrode chamber 14 defines a recess.
  • the open side of the other electrode chamber 14 defines a spigot.
  • the multi-compartment electrolyser apparatus is assembled by placing septa or dividing walls (not shown) between adjacent chambers and inserting tie rods through the holes in each chamber and tightening until the unit is sealed.
  • the septa between the various chambers are isoelectric, buffering membranes, cast onto a support of glass fibres or other suitable material.
  • the membranes are sandwiched between two washers and an O-ring is disposed outside the membrane to provide additional sealing. Such membranes are flow-tight and ensure proper pH control.
  • Figure la illustrates a cap 30 for closing the sample inlets 26.
  • a magnetic stirrer 32 (typically a small teflon coated bar magnet) is suspended from the cap 30 on a wire 33, thread or the like via a swivel joint 34.
  • the length of the wire is such that the stirrer located in the well 20.
  • novel apparatus of the present invention may be used for sub- fractionation of entire cell lysates, tissue extracts, body fluids and of any complex protein/peptide mixture, as a sample treatment, prior to a subsequent two-dimensional analysis step.
  • the membranes at each end of a chambers have pH values encompassing the pis of the proteins to be confined within said chamber.
  • the electrode solutions and sample solutions are added and removed via the sample inlets 26 in the top of each chamber.
  • the sample inlets also allow excess fluid in a particular chamber to escape.
  • the chamber blocks may be made of machined acrylic but will more typically be moulded in a disposable plastic material.
  • the problems of sample carry over from one separation to another would require non- disposable chambers to be scrupulously cleaned after each use and it is more efficient in terms of resources because of the amount of cleaning necessary to simply throw the chamber blocks away after use.
  • the chambers will typically have a volume of 80ml. although smaller volumes, say 5ml could be used.
  • the chamber does not require recirculation of the samples as is the case with existing multi-compartment electrolysers. Instead, the magnetic stirrer moves the sample liquid around the chamber and ensures that the proteins and the like in the sample move past the membranes and also prevents electrodecantion.
  • the manner of mounting the magnetic stirrers in the chamber suspended from the cap means that the stirrers can be easily removed from the chamber for re-use. whilst the chamber can be disposed of.
  • Figure 2 illustrates the use of a plurality of multi-compartment electrolysers 10. as shown in Fig.l, on an electrophoresis platform 50 with an integral power supply, multi-position magnetic stirrer and Peltier cooling. (The floor 52 of the platform is cooled by the Peltier elements)
  • Figure 2 shows the use of two multi-compartment electrolysers 10 on a Peltier cooled multi-position magnetic stirrer platform, it will be clear to those skilled in the art that an electrophoresis platform of this design could be made to contain more or less than 2 multi-compartment electrolysers.
  • Figure 3 illustrates using the same electrophoresis platform 50 as shown in Fig.
  • Figure 4 shows a pi 4.0-5.0 fraction fractionated in the multi-compartment electrolyser shown in Fig. 1.
  • a IPG 3- 10 strip was utilised for the first. IEF dimension, thus demonstrating that indeed, in the 2-D map, only this acidic fraction could be displayed, all other proteins in the remainder of the pH scale having been efficiently removed.
  • Figure 5 shows 2 silver stained pH 3-6 IPGs with whole plasma in the left panel and fractionated plasma in the right panel. The 3 black arrows show matching areas on the 2 gels. The vertical arrow on the right panel indicates pH 5.6 which was the pH of the delimiting membrane between the acidic and albumin chambers.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Environmental & Geological Engineering (AREA)
  • Peptides Or Proteins (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne une nouvelle conception d'électrolyseur à plusieurs compartiments permettant de pré-fractionner des mélanges de protéines complexes, utilisés avant la mise en oeuvre de cartes 2-D. Ces procédés de sous-fractionnement permettent d'éliminer efficacement, via des membranes isoélectriques appropriées à plage étroite, des protéines présentes en grande quantité dans un lysat cellulaire ou dans un fluide corporel. A son tour, le mélange de protéines restant, dépourvu des composants principaux peut être chargé dans une carte 2-D à des niveaux beaucoup plus élevés, garantissant ainsi une capacité de sensibilité et de détection plus grandes des protéines moins abondantes. L'appareil est un électrolyseur à plusieurs compartiments, caractérisé en ce que la recirculation des échantillons n'est pas nécessaire. Cet appareil produit des fractions de protéine totalement compatibles avec les protocoles 2-D subséquents, du fait qu'il utilise une technique de focalisation rendant les échantillons hautement concentrés et dépourvus de sels et de tampons.
PCT/AU2000/001391 1999-11-15 2000-11-14 Appareil d'electrophorese a plusieurs compartiments Ceased WO2001036449A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU12589/01A AU755326B2 (en) 1999-11-15 2000-11-14 Multi-compartment electrophoresis
JP2001538938A JP2003514829A (ja) 1999-11-15 2000-11-14 多室電気泳動
EP00974180A EP1230258A4 (fr) 1999-11-15 2000-11-14 Appareil d'electrophorese a plusieurs compartiments
HK03100703.5A HK1049343A1 (zh) 1999-11-15 2000-11-14 多間隔電泳方法及裝置

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
AUPQ4058A AUPQ405899A0 (en) 1999-11-15 1999-11-15 A method and apparatus for fractionation of complex protein samples in proteome analysis
AUPQ4058 1999-11-15
AUPQ8727 2000-07-12
AUPQ8727A AUPQ872700A0 (en) 2000-07-12 2000-07-12 Electrolyser

Publications (1)

Publication Number Publication Date
WO2001036449A1 true WO2001036449A1 (fr) 2001-05-25

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ID=25646202

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PCT/AU2000/001391 Ceased WO2001036449A1 (fr) 1999-11-15 2000-11-14 Appareil d'electrophorese a plusieurs compartiments

Country Status (4)

Country Link
EP (1) EP1230258A4 (fr)
JP (1) JP2003514829A (fr)
HK (1) HK1049343A1 (fr)
WO (1) WO2001036449A1 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003028857A1 (fr) * 2001-09-28 2003-04-10 Proteome Systems Intellectual Property Pty Ltd Electrolyseur ameliore
WO2003101591A1 (fr) * 2002-06-01 2003-12-11 Novartis Ag Separation de molecules
EP1284809A4 (fr) * 2000-04-18 2004-07-21 Gradipore Ltd Appareil de separation pour petits volumes
US7622028B2 (en) 2003-05-09 2009-11-24 Life Technologies Corporation Solution phase electrophoresis device, components, and methods
US7850835B2 (en) 2003-05-09 2010-12-14 Life Technologies Corporation Solution phase electrophoresis device, components, and methods
US8029658B2 (en) 2004-03-18 2011-10-04 Portmann Instruments A.G. Device and method for filtration and/or separation of molecules, particularly proteins
WO2013067477A1 (fr) * 2011-11-04 2013-05-10 Bio-Rad Laboratories, Inc. Procédés et compositions basés sur l'affinité faisant appel à la régulation électronique du ph
US9234875B2 (en) 2011-11-04 2016-01-12 Bio-Rad Laboratories, Inc. Simultaneous purification of cell components
US9321012B2 (en) 2012-04-04 2016-04-26 Bio-Rad Laboratories, Inc. Electronic protein fractionation
US9658195B2 (en) 2012-02-15 2017-05-23 Bio-Rad Laboratories, Inc. Electronic control of pH and ionic strength
US9766207B2 (en) 2011-11-04 2017-09-19 Bio-Rad Laboratories, Inc. Affinity methods and compositions employing electronic control of pH
CN107246985A (zh) * 2017-06-10 2017-10-13 延边大学 一种利用电泳筛选靶蛋白‑天然产物多组分复合体的方法
WO2018071977A1 (fr) * 2016-10-20 2018-04-26 Memphasys Limited Dispositif d'électrophorèse
WO2020000525A1 (fr) * 2018-06-29 2020-01-02 苏州百源基因技术有限公司 Dispositif de tri de cellules et procédé de tri de cellules

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4644610B2 (ja) * 2006-02-17 2011-03-02 京都市 等電点電気泳動装置
US20120043210A9 (en) * 2007-10-09 2012-02-23 Doucette Alan A Apparatus for Purifying Molecules
JP2012525591A (ja) * 2009-04-27 2012-10-22 プロテイン・デイスカバリー・インコーポレーテツド プログラム可能な電気泳動ノッチフィルターシステムと方法
JP2014059160A (ja) * 2012-09-14 2014-04-03 Sharp Corp 電気泳動用試験具およびその製造方法

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US3533935A (en) * 1968-03-20 1970-10-13 Us Agriculture Liquid zone electrophoresis apparatus
WO1992000795A1 (fr) * 1990-07-07 1992-01-23 Serva Feinbiochemica Gmbh & Co. Dispositif d'electrophorese preparatoire

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Patent Citations (2)

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US3533935A (en) * 1968-03-20 1970-10-13 Us Agriculture Liquid zone electrophoresis apparatus
WO1992000795A1 (fr) * 1990-07-07 1992-01-23 Serva Feinbiochemica Gmbh & Co. Dispositif d'electrophorese preparatoire

Non-Patent Citations (2)

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Title
RIGHETTI P.G. ET AL.: "Preparative protein purification in a multi-compartment electrolyser with immobiline membranes", JOURNAL OF CHROMATOGRAPHY, vol. 475, 1989, pages 293 - 309 *
See also references of EP1230258A4 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1284809A4 (fr) * 2000-04-18 2004-07-21 Gradipore Ltd Appareil de separation pour petits volumes
WO2003028857A1 (fr) * 2001-09-28 2003-04-10 Proteome Systems Intellectual Property Pty Ltd Electrolyseur ameliore
WO2003101591A1 (fr) * 2002-06-01 2003-12-11 Novartis Ag Separation de molecules
US7622028B2 (en) 2003-05-09 2009-11-24 Life Technologies Corporation Solution phase electrophoresis device, components, and methods
US7850835B2 (en) 2003-05-09 2010-12-14 Life Technologies Corporation Solution phase electrophoresis device, components, and methods
US8029658B2 (en) 2004-03-18 2011-10-04 Portmann Instruments A.G. Device and method for filtration and/or separation of molecules, particularly proteins
US9766207B2 (en) 2011-11-04 2017-09-19 Bio-Rad Laboratories, Inc. Affinity methods and compositions employing electronic control of pH
WO2013067477A1 (fr) * 2011-11-04 2013-05-10 Bio-Rad Laboratories, Inc. Procédés et compositions basés sur l'affinité faisant appel à la régulation électronique du ph
US9234875B2 (en) 2011-11-04 2016-01-12 Bio-Rad Laboratories, Inc. Simultaneous purification of cell components
US9658195B2 (en) 2012-02-15 2017-05-23 Bio-Rad Laboratories, Inc. Electronic control of pH and ionic strength
US9321012B2 (en) 2012-04-04 2016-04-26 Bio-Rad Laboratories, Inc. Electronic protein fractionation
WO2018071977A1 (fr) * 2016-10-20 2018-04-26 Memphasys Limited Dispositif d'électrophorèse
EP3528930A4 (fr) * 2016-10-20 2020-05-13 Memphasys Limited Dispositif d'électrophorèse
US11466250B2 (en) 2016-10-20 2022-10-11 Memphasys Limited Electrophoresis device
AU2017344755B2 (en) * 2016-10-20 2023-02-02 Memphasys Limited Electrophoresis device
US12134781B2 (en) 2016-10-20 2024-11-05 Memphasys Limited Electrophoresis device
CN107246985A (zh) * 2017-06-10 2017-10-13 延边大学 一种利用电泳筛选靶蛋白‑天然产物多组分复合体的方法
WO2020000525A1 (fr) * 2018-06-29 2020-01-02 苏州百源基因技术有限公司 Dispositif de tri de cellules et procédé de tri de cellules

Also Published As

Publication number Publication date
EP1230258A4 (fr) 2004-12-22
EP1230258A1 (fr) 2002-08-14
JP2003514829A (ja) 2003-04-22
HK1049343A1 (zh) 2003-05-09

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