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EP2271922A1 - Dispositif, procédé et système de gel pour électrophorèse analytique ou de préparation - Google Patents

Dispositif, procédé et système de gel pour électrophorèse analytique ou de préparation

Info

Publication number
EP2271922A1
EP2271922A1 EP09733965A EP09733965A EP2271922A1 EP 2271922 A1 EP2271922 A1 EP 2271922A1 EP 09733965 A EP09733965 A EP 09733965A EP 09733965 A EP09733965 A EP 09733965A EP 2271922 A1 EP2271922 A1 EP 2271922A1
Authority
EP
European Patent Office
Prior art keywords
gel
electrophoresis
chamber
separating
molecules
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.)
Withdrawn
Application number
EP09733965A
Other languages
German (de)
English (en)
Inventor
Erwin Robert Schmidt
Rudolf Baader
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.)
Johannes Gutenberg Universitaet Mainz
Original Assignee
Johannes Gutenberg Universitaet Mainz
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 Johannes Gutenberg Universitaet Mainz filed Critical Johannes Gutenberg Universitaet Mainz
Publication of EP2271922A1 publication Critical patent/EP2271922A1/fr
Withdrawn legal-status Critical Current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
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional 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
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones

Definitions

  • the present invention relates to an electrophoresis device and a gel system for the analytical or preparative separation of biological molecules, such as nucleic acids or proteins. Furthermore, the invention relates to a method for the isolation of biological molecules from an electrophoresis gel and a method for the electrophoretic separation and collection of biological molecules by a two-dimensional gel electrophoresis.
  • Electrophoresis is still considered to be the most efficient method for the separation of biological molecules, in particular of nucleic acids (eg DNA, RNA) or proteins.
  • An important area of application of electrophoresis is the analysis and preparation of DNA fragments or proteins.
  • carrier materials of separating gels in practice mainly different types of agarose gels (for the separation of nucleic acids) or polyacrylamide gels (for the separation of proteins) are used.
  • the electrophoresis method is also used for the preparation of DNA or proteins via gel extraction methods.
  • the molecules are labeled with special marker substances so that their position, optionally with the aid of physical aids (eg UV light), becomes visible in the gel.
  • the aid of physical aids eg UV light
  • the part of the gel with the molecules contained therein is cut out and the desired molecules are isolated from the gel piece.
  • the process is quite complicated and requires a variety of reagents for its implementation. Further, in the gel extraction method, a considerable part of the sample amount is often lost by the method.
  • two-dimensional gel electrophoresis also called 2D gel electrophoresis
  • 2D gel electrophoresis The combination of two orthogonal electrophoresis steps achieves a high-resolution separation of the individual molecules.
  • rotation of the gel is after the first
  • a device for collecting biological molecules via a preparative electrophoresis device is also described in DE 225 44 89 A1.
  • the device described therein has a funnel-shaped zone for the electrophoresis gel, which at its lower end merges into a narrow opening and communicates with an elution chamber.
  • the elution chamber is provided with an outlet for the electrophoretically separated eluate.
  • this device is not suitable. Furthermore, due to the different migration rate of the molecules, it is difficult to obtain precisely separated fractions.
  • electrophoresis gels are poured between two glass plates which are sealed by two laterally arranged spacers or spacers.
  • Such plate gel systems are used in particular in vertical gel electrophoresis.
  • the gel strength is specified here by the height of the spacer elements.
  • Vertical gel electrophoresis allows a much higher separation of the molecular fractions than, for example, horizontal gel electrophoresis, which is frequently used for the separation of DNA fragments, in which the separating gel (then without plates) lies in a horizontal position in the electrophoresis medium on a slide in the gel chamber.
  • the samples in this chamber must be mixed with a weighting agent (eg glycerin) so that no washout of the samples from the Gelases through the electrophoresis medium.
  • a weighting agent eg glycerin
  • a collecting gel is often used for sample concentration in addition to the separating gel.
  • the present electrophoresis device is characterized in that a sample collector designated here as a collector is used, which consists of one or more juxtaposed sample collecting containers and delimits the separating gel from at least one side.
  • the collector is preferably strip-shaped in shape and at the same time serves as a spacer element for the two gel plates of a gel system of a vertical electrophoresis device.
  • two collectors of the same size as conventional spacers are arranged between the glass plates opposite each other laterally.
  • the individual sample collection vessels of the collector serve to fractionate and collect the electrophoretically separated samples from the separation gel by electrophoretically collecting the molecules into the sample collection vessels.
  • the sample collection containers are cylindrical or cuboid.
  • the method of electrophoretic separation and collection of biological molecules by two-dimensional gel electrophoresis comprising the steps of:
  • the procedure is preferably as follows.
  • the individual sample collection containers are first sealed with liquid gel material at the foot end.
  • the spacer element is expediently placed in a dish with liquid gel material so that the liquid is drawn by cohesive forces into the lumen of the individual sample collecting containers.
  • the sample collection containers are closed at the bottom. The thus sealed collector is then inserted between the two glass plates as a spacer element and the gel is poured as usual between the gas plates.
  • the gel system consisting of separating gel and the two glass plates is inserted into the electrophoresis chamber.
  • guide grooves are preferably formed on the wall of the gel chamber, which have a profile which makes it possible for the gel system to be used in a predetermined orientation.
  • a first electrophoresis step the size-dependent separation of the biological molecules takes place.
  • the invention is not limited to any particular type of biological molecules. All gelelektrophoretisch separable molecules are encompassed by the invention, but in particular nucleic acids such as DNA, RNA or proteins.
  • the first electrophoresis step is carried out until the desired separation of the molecules has taken place. The degree of separation can be monitored by means of a conventional marker in the field.
  • the gel system After performing the first electrophoresis step, the gel system is removed from the gel chamber and rotated through an angle of 90 °, so that the collector is now arranged with the sample collection containers on the underside of the separating gel.
  • the molecules separated in the separation gel by the first electrophoresis step according to their size are allowed to run into the individual sample collection vessels of the collector and collected there.
  • the individual molecules are now fractionated according to their molecular size in the individual sample collection containers.
  • the gel can be removed from the gel chamber and the collected samples isolated from the individual sample collection containers (eg pipetted).
  • This process can also be automated.
  • a capillary is provided in a preferred embodiment, in the hollow cylinder of which an ejection member is arranged, which can be pushed out after being immersed in the sample collecting container of the spacer element that the residual gel after the immersion, which could clog the capillary, are removed.
  • a Multipipettier consisting of several capillaries, all sample collection container of the collector can be treated in a single pipetting step and further processed the samples so removed for analysis or preparation.
  • the collector according to the invention is suitable for both horizontal and vertical Elektrophoresevoriquesen. It is only necessary to ensure that migration of the molecules due to the electric field can take place in the separating gel. In a first step, the size-dependent separation of the molecules takes place and in the second step the collection of the thus separated molecules into the sample collection container of the collector takes place. As a result, the process is suitable for both analytical and preparative separation.
  • FIG. 1 to 3 are isometric views of an embodiment of the electrophoresis device according to the invention.
  • FIG. 4 is a side view of a gel system with the two formed as a collector spacers with sample collection container,
  • FIG. 5 is an isometric view of the gel system shown in FIG. 4; FIG.
  • FIG. 6 shows an embodiment of the chamber bottom of the gel chamber
  • Fig. 7 shows an embodiment of the guide channel for the gel system
  • FIG. 8 shows a further embodiment of the electrophoresis chamber according to the invention for the separation of proteins. Ways to carry out the invention and commercial usability:
  • a first embodiment of the electrophoresis chamber according to the invention is shown. Visible is the gel chamber 10 and the gel system used therein, consisting of a separating gel 3, a glass plate as a front plate 24 and a glass plate as back plate 26. As laterally delimiting spacer elements according to the invention designed as a collector individual sample collection container 22 can be seen directly to the separating gel 3 adjacent.
  • the gel chamber 10 is filled with electrophoresis medium 20 for operation and covered with a cover 8 for protection. Power is supplied via the electrical connections 12, 14.
  • the electrical contact elements 16, 18 (conductor wires) for establishing an electric field for the electrophoresis are arranged in the two chamber halves of the gel chamber 10.
  • the positive contact wire in the front half of the chamber in the region of the chamber bottom 6 (contact element 16).
  • the contact element 16 is protected on the side with an insulating tube 21.
  • the contact element 18 is arranged in the region of the chamber ceiling.
  • FIG. 2 shows an isometric view of the electrophoresis device shown in FIG.
  • the gel system consisting of faceplate 24, backplate 26 and separator gel 3 disposed therebetween is removed from the gel chamber and rotated through an angle of 90 °, so that the sample collection container 22 of the spacer disposed on the underside of the separation gel 3 are.
  • a guide groove 15 formed on the wall of the gel chamber 10 is provided. The guide channel 15 separates the gel chamber 10 into two comb halves, in which the electrical contact elements 16, 18 are arranged as described above.
  • FIG. 3 shows a top view of the electrophoresis device according to the invention.
  • the gel system is inserted into the guide channel 15 of the gel chamber 10.
  • Fig. 4 the gel system according to the invention is shown in more detail.
  • This consists of the two glass plates 24, 26 and the interposed separating gel 3.
  • a sample application bag 25 can be seen for the sample application.
  • the two glass plates 24, 26 are held together by connecting means 23.
  • the collector with the sample collection containers 22 also serves as a spacer for the two glass plates 24, 26.
  • the sample collection containers 22 are sealed at their base with gel material 28 to prevent diffusion of molecules from the containers during the second electrophoresis step.
  • two spacer elements are provided with sample collection containers 22, which are arranged opposite each other and limit the separating gel 3 on both sides.
  • Fig. 5 shows a preferred variant of the gel system according to the invention, in which the front plate 24 has a smaller diameter than the back plate 26.
  • Fig. 6 it is shown how the glass plates 24, 26 are used to the chamber bottom 6.
  • the different sized glass plates 24, 26 have been found to be advantageous to produce a directed electric field through the separating gel 3.
  • 6 is in the chamber bottom in the
  • Chamber half with the smaller front plate 24, a retaining plate 27 is provided, which closes the otherwise open separating gel 3 at the bottom laterally.
  • FIG. 7 shows a further preferred embodiment of the electrophoresis device according to the invention.
  • the two glass plates 24, 26 may be inserted into the gel chamber 10 in a predetermined orientation.
  • the guide groove 15 is designed such that the orientation when inserting the gel system is predetermined if the two glass plates 24, 26 are of different sizes.
  • FIG. 8 shows a further embodiment of the electrophoresis device according to the invention. This is a chamber for separation of proteins by SDS-PAGE.
  • the vertical arrangement achieves high resolution of the molecules to be separated.
  • the separation principle is similar to the method described above.
  • the spacer elements according to the invention with the sample collection containers 22 can be used in any conventional gel chamber. It is only necessary to ensure that a directed flow of current through the separating gel 3 takes place. Further embodiments of the electrophoresis device according to the invention will become apparent to the person skilled in the art with reference to the present invention description.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un dispositif d'électrophorèse constitué d'une chambre à gel (10) destinée à contenir un fluide d'électrophorèse (20); d'un système de gel disposé dans la chambre à gel (10) et apte à en être prélevé, contenant un gel de séparation (3) pour la séparation par électrophorèse de molécules biologiques, par exemple des acides nucléiques ou des protéines; d'éléments (16, 18) de contact électrique qui produisent un champ électrique à travers le gel de séparation (3) et éventuellement un couvercle (8) destiné à être placé sur la chambre à gel (10), ledit dispositif étant caractérisé en ce que le gel de séparation (3) est délimité sur au moins un côté par un élément d'écartement configuré comme collecteur qui comprend plusieurs récipients (22) de collecte d'échantillons juxtaposés pour le fractionnement et le recueil des molécules séparées par électrophorèse. L'invention concerne en outre un procédé de séparation et de recueil par électrophorèse de molécules biologiques par électrophorèse bidimensionnelle sur gel.
EP09733965A 2008-04-24 2009-04-20 Dispositif, procédé et système de gel pour électrophorèse analytique ou de préparation Withdrawn EP2271922A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102008020428A DE102008020428B3 (de) 2008-04-24 2008-04-24 Vorrichtung und Verfahren und Gelsystem zur analytischen und präparativen Elektrophorese
PCT/EP2009/002852 WO2009129972A1 (fr) 2008-04-24 2009-04-20 Dispositif, procédé et système de gel pour électrophorèse analytique ou de préparation

Publications (1)

Publication Number Publication Date
EP2271922A1 true EP2271922A1 (fr) 2011-01-12

Family

ID=40758683

Family Applications (1)

Application Number Title Priority Date Filing Date
EP09733965A Withdrawn EP2271922A1 (fr) 2008-04-24 2009-04-20 Dispositif, procédé et système de gel pour électrophorèse analytique ou de préparation

Country Status (4)

Country Link
US (1) US20110114487A1 (fr)
EP (1) EP2271922A1 (fr)
DE (1) DE102008020428B3 (fr)
WO (1) WO2009129972A1 (fr)

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EP2470691B1 (fr) 2009-08-24 2014-12-03 Life Technologies Corporation Cassette et peigne d'électrophorèse sur gel
USD794823S1 (en) 2010-08-24 2017-08-15 Life Technologies Corporation Electrophoresis tank with a base and cassette inserted in
USD719277S1 (en) 2010-08-24 2014-12-09 Life Technologies Corporation Electrophoresis wedge-well comb
USD667134S1 (en) 2011-04-01 2012-09-11 Life Technologies Corporation Electrophoresis apparatus
USD698037S1 (en) * 2012-06-01 2014-01-21 Life Technologies Corporation Electrophoresis tank
US9599590B2 (en) 2012-10-12 2017-03-21 Sage Science, Inc. Side-eluting molecular fractionator
CA2964487C (fr) 2014-10-15 2024-03-19 Sage Science, Inc. Appareils, procedes et systemes pour le traitement automatise d'acides nucleiques et la preparation electrophoretique d'echantillons
USD757958S1 (en) * 2014-12-03 2016-05-31 Edvotek Inc. Electrophoresis apparatus
USD806894S1 (en) 2015-03-19 2018-01-02 Life Technologies Corporation Electrophoresis comb with wedge-shaped teeth
CN108699101B (zh) 2015-11-20 2022-04-19 塞奇科学股份有限公司 用于基因组dna片段的靶向纯化的制备型电泳方法
AU2018250330A1 (en) 2017-04-07 2019-09-19 Sage Science, Inc. Systems and methods for detection of genetic structural variation using integrated electrophoretic DNA purification
JP2023534015A (ja) 2020-07-13 2023-08-07 ライフ テクノロジーズ コーポレーション 電気泳動及び電気泳動転写デバイス、システム、並びに方法
USD975873S1 (en) * 2020-07-13 2023-01-17 Life Technologies Corporation Electrophoresis and electrotransfer device
CN114965657B (zh) * 2022-06-07 2023-11-24 苏州科技大学 一种生物电泳聚合物制胶自动化系统及装置

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US5041203A (en) * 1988-06-28 1991-08-20 The University Of Texas System Apparatus and procedure for rotating gel electrophoresis
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Also Published As

Publication number Publication date
WO2009129972A1 (fr) 2009-10-29
DE102008020428B3 (de) 2009-07-16
US20110114487A1 (en) 2011-05-19

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