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WO2006036119A1 - Procede et dispositif pour focalisation isoelectrique - Google Patents

Procede et dispositif pour focalisation isoelectrique Download PDF

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Publication number
WO2006036119A1
WO2006036119A1 PCT/SE2005/001427 SE2005001427W WO2006036119A1 WO 2006036119 A1 WO2006036119 A1 WO 2006036119A1 SE 2005001427 W SE2005001427 W SE 2005001427W WO 2006036119 A1 WO2006036119 A1 WO 2006036119A1
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WO
WIPO (PCT)
Prior art keywords
gel
channels
ipg
gels
strip
Prior art date
Application number
PCT/SE2005/001427
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English (en)
Inventor
Bengt Bjellqvist
Original Assignee
Ge Healthcare Bio-Sciences Ab
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 Ge Healthcare Bio-Sciences Ab filed Critical Ge Healthcare Bio-Sciences Ab
Publication of WO2006036119A1 publication Critical patent/WO2006036119A1/fr

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Classifications

    • 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
    • C07K1/28—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
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing

Definitions

  • the present invention relates to the field of electrophoresis and more specifically to a method and device of separating protein and/or peptide components by isoelectric focusing by arranging the gels or strips, preferably IPG (immobilised pH gradient) gels or strips, in a novel way during focusing.
  • the gels or strips are arranged with the width of the gels in substantially vertical direction.
  • biomolecules such as proteins and peptides
  • Some biomolecules need to be isolated as a last step of a biotechnological method for the production thereof, for example in the preparation of protein-based pharmaceutical compounds.
  • biomolecules for analytical purposes in order to be able to quantitate and identify the proteins and/or peptides present in a sample.
  • Electrophoretic methods are commonly used in the separation step. A wide variety of methods are used for the detection and quantification of the separated proteins. For identification and characterisation of separated proteins MS methods are normally used as these methods are fast and require very small amounts of proteins and/or peptides.
  • electrophoresis involves the movement of charged particles or ions in an electric field.
  • the driving force for the electrophoretic transport of an ion or a particle is the product of the effective charge of the particle and the potential gradient, and the frictional resistance of the medium balances this force.
  • zone electrophoresis Based on how the pH and ionic strength is established along the separation distance, basically three different types of electrophoretic methods can be distinguished: zone electrophoresis, isoelectric focusing and isotachophoresis.
  • isoelectric focusing In isoelectric focusing (IEF), the separation takes place in a stationary pH gradient that occupies the whole separation distance and is arranged so that the pH in the gradient increases from anode towards the cathode. While other alternatives also exist, the pH gradients required in isoelectric focusing are in practice generated in two different ways: with the aid of a solution of carrier ampholytes or with an immobilised pH gradient.
  • IPG immobilised pH gradient
  • the immobilised charged or chargeable groups used are normally a limited number of carboxylic groups or amino groups with different pK-values distributed within or close to the pH gradient, which is to be generated.
  • concentration of the charged or chargeable groups is varied along the separation distance in a manner causing the pH at which the wall or the gel matrix has a zero net charge to increase from the anode to the cathode.
  • a commercially available example of a system for generation of immobilised pH gradients is the lmmobiline Il systemTM (Amersham Biosciences, Uppsala, Sweden), wherein a pH gradient covalently attached to a polyacrylamide gel is formed.
  • Immobilised pH gradients are truly stationary and today they are normally used together with carrier ampholytes. In this combination the immobilised gradient determine the resulting pH gradient, while the carrier ampholytes contribute with conductivity.
  • the length of the application zone is not critical in isoelectric focusing.
  • the sample can be mixed into the separation medium and at the start of the separations be present all along the separation distance, but for analytical applications the sample is normally applied close to either the anode or the cathode.
  • sample volumes are usually applied in sample cups but when larger volumes are used the sample is included in the solution used for rehydration of the IPG strip and/or added to large filter paper positioned between the end of the IPG- strip and either the anodic or cathodic electrode so called "paper bridge" application (Sabounchi-Schutt F, Astrom J, Olsson I, Eklund A, Grunewald J, Bjellqvist B. An immobiline DryStrip application method enabling high-capacity two-dimensional gel electrophoresis.Electrophoresis. 2000 Nov;21(17):3649-56).
  • capillaries and different types of gels are used also in isoelectric focusing .
  • wet gels ready to use are Ampholine PAGplateTM gels, which exist for a number of pH ranges pH3.5-9.5, pH 4.0-6.5, pH 5.5- 8.5 and pH 4.0-5.0.
  • dry gels are Clen Gel IEFTM and Immobiline Dry PlateTM gels.
  • lmmobiline DryStripTM gels A special variant of the latter type is the lmmobiline DryStripTM gels, which are designed to be used as first dimension in two-dimensional electrophoresis.
  • the exuded liquid originally contains protein and if the liquid remains in contact with the strip additional proteins will diffuse from the strip out to this liquid.
  • the spreading of the liquid along the strip will result also in protein transport and 2-D maps showing extensive horisontal streaking.
  • Streaking connected to liquid exudation can also be caused by electrosmosis generated by the transport of hydrogen ions, hydroxyl ions, buffer ions or proteins in a strip containing an immobilised pH gradients.
  • an lmmobiline DryStripTM containing a pH gradient pH 4-5 the presence of hydrogen ions transported towards the cathode will cause electrosmotic pumping of liquid towards the cathode and exudation of liquid at or close to the cathode.
  • a filter paper placed between the electrode and the IPG-strip is normally used to absorb exuded liquid. If the sample added to the strip contains large amounts of ions transported to the cathode or if very long focusing times are required spreading of this liquid along the IPG strip can still not be avoided unless the paper is exchanged a couple of times during the focusing.
  • the sample contains large amount of an amphoteric substances, such as glycine or other amino acids or alternatively a Good ' s buffers like MOPS or HEPES which focus within the pH range covered by the IPG strip, water will accumulated at the isoelectric point and exudation and streaking will take place and the same thing will happen if the sample contains large amounts (in the region 10 to 100 ug) of an individual protein focusing within the pH region.
  • an amphoteric substances such as glycine or other amino acids or alternatively a Good ' s buffers like MOPS or HEPES which focus within the pH range covered by the IPG strip
  • IPG strips are placed on a flat horizontal surface with the plastic foil in contact with a cooled bottom surface.
  • IPG strips are placed on a flat horizontal surface with the gel side in contact with a cooled bottom surface, such as in US 6 113 766 (IPGphor).
  • 4/ IPG strips are according to WO 02/ 070111 placed on the surface of a rotating drum.
  • the IPG strips contain preformed pH gradients covering different pH intervals, such as 3-5-6, 5.3-6.5, 6.2-7.5, 7-11 , but also in broader intervals like pH 3-11.
  • the resolution resulting in an isoelectric focusing experiment increases with decreased pH gradient slope, dpH/dx.
  • the resolution given with a broad gradient, such as 3-10 in a 24 cm long IPG strip is inadequate and focusing in narrow pH range strips is required.
  • a problem appearing in this context is that very frequently the sample amounts available are limited and not large enough to allow sample application on 3-4 narrow range strips.
  • WO 02/ 070111 also describes a method for manufacture of long IPG gels.
  • the gel is placed on a rotating drum. This technique avoids some of the above drawbacks with long IPG gels but is complicated and needs special equipment.
  • the present invention provides a method of running isoelectric focusing which avoids liquid exudation resulting in spreading of protein along the gel strip within which the focusing is done. Furthermore, the invention provides a simplified way of running long gels or strips with high resolution. The method enables focusing of proteins and peptides in sharp spots.
  • the invention also provides a device for use in said method.
  • the device should be adjusted for IEF with both short and long strips or gels.
  • the device should be designed to avoid liquid exudation spreading in the direction of the pH gradient on the gel or strip.
  • the liquid exuded should not be allowed to be in contact with the gel.
  • the invention relates to a method of separating proteins/peptides in a biological mixture by isoelectric focusing (IEF) using gel(s) or strips, preferably immobilised pH gradient (IPG) gel(s) or strip(s), comprising the following steps: a) placing the gel(s) in channel(s) of a focusing unit with the width of the gel(s) arranged substantially vertically in said channel(s) and applying sample to the gel(s) either before and/or after placing the gels in the focusing unit; b) positioning of electrodes on said gel(s), wherein a cathode is placed at the basic end and an anode at the acidic end of each of said gel(s); and c) applying a voltage between said cathode(s) and anode(s), wherein, during focusing, any exudates formed from the gel(s) is drained off from the gel.
  • IPG immobilised pH gradient
  • IPG gels of any length, such as 7-24 cm or longer.
  • Substantially vertical means in this context that it is substantially perpendicular to the direction of focusing, i.e. to the direction of the pH-gradient of the IPG gel.
  • a low viscosity paraffin or silicone oil is added to cover the gel(s) and electrodes after step b). If no oil is added, the method may be run in a closed system. Oil or a closed system is needed because of the high voltages applied but also as a mean to avoid evaporation of water and crystallisation of urea in the IPG strips.
  • the IPG gel is at least 30 cm long, preferably at least 50 cm long and more preferably at least 90 cm long, preferably 96 cm.
  • the gel is 96 cm it can be divided into 4 x 24 cm after the run and this length is adopted to the second dimension of 2D electrophoresis.
  • the gel or strip may also be divided into two or more sections before IEF which are connected to each other, via for example polyacrylamide bridges as described in WO 05/026715. In this way, serial IEF may be run.
  • the channels are curve shaped.
  • the channels are spiral formed.
  • the pH-gradient of the IPG gel is optional.
  • a broad pH range will be used, such as pH 3-10 or 3-11 or 3-12.
  • the gel(s) or strip(s) may be rehydrated before or after it/they is/are placed in said channel(s).
  • excess liquid is allowed to drain off the gel(s) into the bottom of the channel(s) in such a way that the gel(s) no longer is/are in contact with said liquid. This is a great advantage of the invention since no excess liquid from the gel will interfere with and disturb the separation process.
  • IPG strips have the capability of resolving proteins in a broad pH range due to the simple fact that they are long and thereby the distance between focused spots with different pH becomes larger.
  • a organic disulfide such as DeStreakTM is present in the rehydration solution.
  • DeStreakTM prevents streaking of the proteins and peptides and thereby gives more distincly focused spots.
  • At least two IPG gels may be placed in parallel but insulated from each other in separate channels in the focusing unit.
  • the invention in a second aspect, relates to a device for use in the above method comprising a focusing unit (1) provided with curve-shaped channel(s) (2) and electrodes (3).
  • the channels are spiral formed and have a length of at least 30 cm.
  • the channels have a width of 1-10 mm, preferably 3-6 mm, and a depth of 7-15 mm.
  • the device is adjusted for IEF running of long IPG strips or gels by arranging the strips or gels spirally in the device with the gel width in a substantially vertical direction.
  • a ridge or shelf is provided in the channels to prevent the gels from sliding down to the bottom of said channels and to allow liquid to drain off to a bottom space in the channels.
  • the ridge or shelf may be divided into separate sections and may be in the form of distance laths.
  • at least two channels are arranged in parallel in said focusing unit.
  • 1-6 gels can be arranged in 1-6 channels in the device.
  • the number of electrode pairs (anode and cathode) in the device corresponds to the number of gels to be run.
  • the outer electrode(s) should preferably be movable for adjustment to gel(s) of different length.
  • the inner electrode(s) may be permanently arranged.
  • the device according to the invention may be a separate unit or may be integrated in a multipurpose unit, like a MultiphorTM, wherein said focusing unit is provided with an inlet and an outlet for a cooling liquid under and/or between the channels.
  • a lid may be arranged over the focusing unit.
  • the device of the invention comprises channels in which the gel strips to be focused are placed in horizontal positions and with the plastic foil supporting the gel strip towards one of the vertical walls forming the channel within which the specific strip is placed with the width of the gel substantially in a vertical direction.
  • the channel wall could be straight but also slightly curved representing a part of a wall in a cylinder or tubing.
  • the channels could be straight but in reality a curved form is preferred as this form make it easier to ascertain good contact between the vertical wall and the film supporting the gel or strip.
  • the channels should contain the arrangements necessary for establishing contact between the two ends of the IPG strips and an anodic and cathodic electrode respectively, and at each end of the strips there should be the space and arrangements required to allow for anodic and/or cathodic paper bridge application.
  • the width of the channels could correspond to the thickness of the rehydrated IPG-strips including the contribution from the support film in which case the IPG strips are pressed (pushed) down towards the bottom of the channel. In this case the channels are possible to use as well for the re-swelling as for the focusing of the IPG strips.
  • the width of the channels exceeds the thickness of IPG strips in which case a minimum distance of 2 mm is maintained between the bottom of the channels and the IPG strips either with the aid of distance laths or by placing the strip on a shelf where said shelf follows the vertical wall towards which the strip is placed and where the width of said shelf does not exceed the thickness of the IPG strip.
  • the arrangement is made in order to allow exuded liquid to drain off the strip in a direction perpendicular to the pH gradient with minimal spreading along the gel in the direction of the pH gradient and to accumulate in a volume beneath the strip which is large enough to ascertain that there will be no contact between exuded liquid and IPG strip.
  • the minimum depth of the channel should allow the IPG strip to be covered with oil during focusing and result in a minimum distance between the oil-air interface and the IPG strips of 2 mm and preferably this distance should be somewhere between 3 and 6 mm.
  • the above preferred form is provided with curve-shaped or spiral formed channels. These channels optimize the contact between the plastic film and the vertical wall of the channel.
  • the spiral formed channel is especially advantageous for running of long IPG strips since the spiral form requires a minimum of space.
  • Fig. 1 shows a schematic top view of device with spiral formed channels for IEF running of long IPG strips.
  • Fig. 2 is a cross section along line A-A in Fig. 1 and show how the gels and electrodes are arranged in the channels.
  • a gel is cast between two glass plates of about 100 cm length, 24 cm breadth and 0.5 mm thickness, wherein the glass plates are sealed along the sides and the bottom.
  • a GelbondTM or other gel adhering film is tightly arranged on one of the glass plates.
  • a gradient is formed by using a heavy solution of acrylamide/bis acrylamide + 20% glycerol and a light solution of acrylamide/bis acrylamide without glycerol. At the same time a gradient is formed by using a mixure of acid immobilines and basic immobilines, respectively.
  • the gradient is from 20%-0% glycerol and preferably from pH 3-10, - 11 or -12.
  • TEMED and ammonium persulphate is added to start the polymerisation reaction.
  • the gradient starts with only heavy solution and only acid immobilines.
  • the highest concentration of glycerol will be at the bottom of the gel as well as the most acid pH- value.
  • the IPG gel is allowed to polymerise for 1-2 hours.
  • the GelbondTM films with adhered IPG gels are removed from the glass plates and excess of non-polymerised material is washed off.
  • the GelbondTM films with adhered IPG gels are dried in an oven fur sufficient time to dry the gel on the film. Finally, the film is cut into long narrow (0.2-1 cm) strips.
  • the IPG strips are rehydrated into a correct size. If they are rehydrated into a size that is too large, too much water will be comprised in the gel and this is not good because during focusing this excess liquid will be squeezed out of the strip together with proteins. If the strips are insufficiently rehydrated, then the time required for focusing will increase due to decreased protein mobilities. Normally the resulting rehydration volume is limited by the amount of liquid added, but it could also be determined through geometrical constraints of the chamber used for the rehydration
  • the solution used in the rehydration step could have varying composition but normally the solution contains 8-9 M urea or alternatively a mixture of urea and thiourea, some type of non ionic or amphoteric detergent like CHAPS or Triton-X100, carrier ampholyte and either a reducing agent like DTT or an organic disulfide (DeStreakTM) .
  • the rehydration is done for a minimum of 6 hours and normally over-night (14-20 hours). To avoid evaporation and urea crystallisation, protection against evaporation is needed. This can be achieved by covering the gel strip with paraffin or silicone oil and/or with a lid enclosing the chamber.
  • the rehydration may be made in the device of the invention in which case the device will be sized for that purpose.
  • the strip is placed on its edge (i.e. with its width in a vertical position) for focusing as shown in the cross section of Fig. 2.
  • the strip is spirally arranged in the channel of the device of the invention.
  • the strip is rehydrated in the device but may also be rehydrated in a preceding step.
  • the device is placed on a MultiphorTM.
  • Sample is added to the strip by either rehydration or paper bridge application.
  • a positively charged paper may be used such as described in co-pending application SE 0303581-3.
  • Anode and cathode electrodes are placed at each end of the strip.
  • Low viscosity paraffin or silicone oil is added to cover the strips, the lid is closed and a voltage is applied.
  • the voltage profile is as common practice, i.e. first a very low voltage to run in the sample and then higher voltages.
  • the vertical positioning of the strips in the channels allows excess liquid formed during the run to flow into the bottom of the channel without contact with the gel.
  • the device is built for voltages of 10-10O kV.
  • Fig. 2 shows the gel in solid black and the end of an electrode placed in the channels. If the sample is applied by paper bridge application, then a not shown filter paper will be located between the gel and electrode.
  • the device of the invention offers easy handling and requires a minimum of space for rehydration and IEF running of long IPG gels.
  • the IEF run may be used as a pre-treatment step before 2D electrophoresis or other separation.
  • the high resolution in the IEF run according to the invention enables analysis of low abundant proteins.
  • the proteins in the strips may alternatively be directly further separated in the second electrophoresis step of 2D electrophoresis or may be directly analysed.
  • the strips may be cut into suitable lengths before 2D electrophoresis or other analysis.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Peptides Or Proteins (AREA)
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Abstract

Cette invention relève du domaine de l'électrophorèse et concerne plus spécifiquement un procédé et un dispositif permettant de séparer des composants protéiques et/ou peptidiques par focalisation isoélectrique en disposant les gels ou bandes, de préférence un ou plusieurs gels ou bandes à gradient de pH immobilisé (IPG), d'une manière innovante pendant la focalisation. Les gels ou bandes sont disposés de façon que la largeur des gels soit orientée substantiellement à la verticale. Cet agencement occupe un minimum d'espace pour de longs gels IPG et évite que du liquide ne se répande sur le gel pendant les longues périodes de focalisation. La focalisation isoélectrique appliquée dans le dispositif de cette invention permet d'obtenir une haute résolution de protéines et d'effectuer l'analyse de protéines peu abondantes.
PCT/SE2005/001427 2004-09-30 2005-09-28 Procede et dispositif pour focalisation isoelectrique WO2006036119A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE0402375A SE0402375D0 (sv) 2004-09-30 2004-09-30 Method and device for isoelectric focusing
SE0402375-0 2004-09-30

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WO2006036119A1 true WO2006036119A1 (fr) 2006-04-06

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007126354A1 (fr) * 2006-04-27 2007-11-08 Ge Healthcare Bio-Sciences Ab Pont d'électrodes

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6113766A (en) * 1997-06-09 2000-09-05 Hoefer Pharmacia Biotech, Inc. Device for rehydration and electrophoresis of gel strips and method of using the same
US20020100690A1 (en) * 2000-09-29 2002-08-01 Ben Herbert Electrophoresis system
WO2002092200A1 (fr) * 2001-05-10 2002-11-21 Invitrogen Corporation Procedes et appareil d'electrophorese de milieux de separation hydratables avant le coulage
EP1353171A2 (fr) * 2002-04-12 2003-10-15 Tecan Trading AG Porte-bande, chambre, cassette et procédé d'électrophorèse 2-D

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6113766A (en) * 1997-06-09 2000-09-05 Hoefer Pharmacia Biotech, Inc. Device for rehydration and electrophoresis of gel strips and method of using the same
US20020100690A1 (en) * 2000-09-29 2002-08-01 Ben Herbert Electrophoresis system
WO2002092200A1 (fr) * 2001-05-10 2002-11-21 Invitrogen Corporation Procedes et appareil d'electrophorese de milieux de separation hydratables avant le coulage
EP1353171A2 (fr) * 2002-04-12 2003-10-15 Tecan Trading AG Porte-bande, chambre, cassette et procédé d'électrophorèse 2-D

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007126354A1 (fr) * 2006-04-27 2007-11-08 Ge Healthcare Bio-Sciences Ab Pont d'électrodes
US7964075B2 (en) 2006-04-27 2011-06-21 Ge Healthcare Bio-Sciences Ab Electrodic bridge

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