WO2005098408A1 - Electrophorese en gel multifonctionnelle et appareil - Google Patents

Electrophorese en gel multifonctionnelle et appareil Download PDF

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Publication number
WO2005098408A1
WO2005098408A1 PCT/US2005/011091 US2005011091W WO2005098408A1 WO 2005098408 A1 WO2005098408 A1 WO 2005098408A1 US 2005011091 W US2005011091 W US 2005011091W WO 2005098408 A1 WO2005098408 A1 WO 2005098408A1
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WO
WIPO (PCT)
Prior art keywords
cassette
tank
gel
lid
buffer
Prior art date
Application number
PCT/US2005/011091
Other languages
English (en)
Inventor
Charles B. Scott
John Victor Mcknight
Steven J. Elliott
Original Assignee
C.B.S. Scientific Co., Inc.
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 C.B.S. Scientific Co., Inc. filed Critical C.B.S. Scientific Co., Inc.
Publication of WO2005098408A1 publication Critical patent/WO2005098408A1/fr

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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
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44739—Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots

Definitions

  • the present invention relates to the field of electrophoretic separation of molecules, and, more particularly, to designs for slab gel electrophoresis and electroblotting apparatus.
  • Electrophoresis is the process of separating molecules on the basis of their migration through a medium in an applied electric field.
  • a molecule will migrate towards the pole (cathode or anode) that carries a charge opposite to the net charge carried by the molecule.
  • This net charge depends in part on the pH of the medium in which the molecule is migrating.
  • One common electrophoretic procedure is to establish solutions having different pH values at each end of an electric field, with a gradient range of pH in between. At a certain pH, the isoelectric point of a molecule is obtained and the molecule carries no net charge. As the molecule crosses the pH gradient, it reaches an isoelectric point and is thereafter immobile in the electric field.
  • Electrophoresis in a polymeric gel adds two advantages to an electrophoretic system.
  • t e polymeric gel stabilizes the electrophoretic system against convective disturbances.
  • the gel provides a porous passageway through which the molecules must travel. Since larger molecules will travel more slowly through the passageways than smaller molecules, use of a gel permits the separation of molecules by both molecular size and isoel&ctric point.
  • Both vertical and horizontal assemblies are routinely used in gel electrophoresis. In a vertical apparatus, the sample wells are formed in the same plane as the gel and are loaded vertically.
  • a horizontal gel will generally be open on its upper surface, and the sample wells are formed normal to the plane of the gel and also loaded vertically. Electrophoresis in slab gels offers versatility and speed to the laboratory technician performing analyses of biological samples.
  • a single slab gel can serve as the separation medium in which a large number of individual samples can be analyzed simultaneously by dividing the slab into parallel lanes and using one lane for each sample. This affords not only speed and an efficient use of labor, energy, materials, equipment, and time, but also eliminates many of the problems that commonly arise when separate procedures are performed on each of a series of samples, the problems including for example nonuniformity that arises from variations in gel quality and operating conditions and the risk of operator error.
  • slab gels are used in the performance of two- dimensional electrophoresis, in which a first dimension separation is performed in a linear medium such as a gel tube or strip, which is then placed along one edge of the slab for a second dimension separation in a direction transverse to the axis of the linear medium.
  • a first dimension separation is performed in a linear medium such as a gel tube or strip, which is then placed along one edge of the slab for a second dimension separation in a direction transverse to the axis of the linear medium.
  • a first dimension separation is performed in a linear medium such as a gel tube or strip, which is then placed along one edge of the slab for a second dimension separation in a direction transverse to the axis of the linear medium.
  • a first dimension separation is performed in a linear medium such as a gel tube or strip, which is then placed along one edge of the slab for a second dimension separation in a direction transverse to the axis of the linear medium.
  • two-dimensional electrophoresis one of the most common
  • a gel electrophoresis system is typically set up as follows: A gel-forming solution is allowed to polymerize between two glass plates that are held apart on two sides by spacers. These spacers determine the thickness of the gel.
  • sample wells are formed by inserting a comb-shaped mold into the liquid between the glass plates at one end and allowing the liquid to polymerize around the mold.
  • the gel may be cast with a flat top and a pointed comb inserted between the plates so that the points are slightly imbedded in the gel.
  • Small, fluid-tight areas between the points can be filled with a sample.
  • the top and bottom of the polymerized gel are placed in electrical contact with two separate buffer reservoirs. Molecule samples are loaded into the sample wells via a sample- loading implement inserted between the two glass plates and the sample is injected into the well. To prevent sample mixing, it is advantageous to inject the sample as close to the gel as possible. It is difficult to place the tip of the pipette or loading implement close to the gel because the pipette tip is often wider than the gel. An electric field is then set up across the gel, and the molecules begin to move into the gel and separate according to their size. Once the electrophoretic separation process is complete, the slab gel is usually subject to further processing in order to analyze and store the results.
  • the size-sorted molecules an be visualized in several ways. After electrophoresis, the gels can be bathed in a molecule-specific stain, which renders the groups of size-sorted molecules visible to the eye. For greater resolution, the molecules can be radioactively labeled and the gel exposed to X-ray film. The developed film indicates the migration positions of the labeled molecules.
  • the slab gel can be subjected to a blotting procedure, such as electroblotting (known generally as Western Blotting), in order to migrate the separated molecules onto a piece of blotting paper, which is easier to handle for analysis, and more stable for storage and later reference.
  • electroblotting known generally as Western Blotting
  • the present invention provides a system for performing gel electrophoresis and electroblotting.
  • the invention provides apparatus comprising a tank with a removable lid, a cassette support assembly adapted to fit into the tank and allow the lid to mount on the tank, the support assembly comprising retaining means for holding at least one slab gel cassette or one electroblotting cassette in a substantially vertical orientation, where the retaining means will substantially seal the cassette against the support to substantially prevent buffer fluid leakage therebetween.
  • a cassette mounting and securing means for mounting the cassette support in the tank such that any cassettes held by the support are substantially vertically oriented, and releasably secured to the cassette support; and first and second electrodes arranged to impose an electric potential substantially uniformly across said cassette.
  • a further aspect of the invention provides a temperature adjustment system for adjusting the interior temperature of the tank while the apparatus is in operation.
  • Figure 1 is an exploded perspective view of a complete assembly of one embodiment of the present invention
  • Figure 2 is an exploded perspective view of the core/cassette components of the assembly of Figure 1
  • Figure 3 A-D presents a perspective view, a side elevation, a horizontal front elevation and a top plan view of the core assembly component of the assembly of Figure 1
  • Figure 4A-D presents a perspective view, a side elevation, a horizontal front elevation and a top plan view of the support stand component of the assembly of Figure 1
  • Figure 5 presents a perspective view of a complete gel core assembly component of the present invention
  • Figure 6 is a detail side elevation of the latch portion of the assembly of Figure 5
  • Figure 7 is a perspective view with portions broken away of a tank of the invention according to Figure 1
  • Figure 8A-D presents a perspective view, a side elevation, a horizontal front elevation and a top plan view of a blotting cassette of the invention
  • Figure 9 is a perspective view of an open blotter cassette of Figure 8
  • Figure 10 is
  • the present mvention comprises an apparatus for performing various types of electrophoretic and other electro-induced separation processes. As is shown in the drawings attached hereto, embodiments of the present apparatus contain a number of improvements and advantageous features compared to previously known devices.
  • the present mvention provides a system for performing gel electrophoresis and electroblotting.
  • the invention provides apparatus comprising a tank with a removable lid, a cassette support assembly adapted to fit into the tank and allow the lid to mount on the tank, the support assembly comprising retaining means for holding at least one slab gel cassette or one electroblotting cassette in a substantially vertical orientation, where the retaining means will substantially seal the cassette against the support to substantially prevent buffer fluid leakage therebetween.
  • a cassette mounting and securing means for mounting the cassette support in the tank such that any cassettes held by the support are substantially vertically oriented, and releasably secured to the cassette support; and first and second electrodes arranged to impose an electric potential substantially uniformly across said cassette.
  • a vertical slab gel device should be understood in general terms.
  • the various components of such a device generally include a support framework with an upper electrolyte or buffer reservoir, a pair of apposing plates, typically glass plates, into which a gel has been cast, and a container, or tank, into which the device is placed, and which serves as the second reservoir.
  • the gap between the two plates is set by a pair of spacers positioned along the lateral edges of the plates, and the slab gel occupies a portion of the gap.
  • the plates and spacers will either be of unitary construction formed by molding or welding, or held together by clamps of conventional construction well known to those skilled in the manufacture or use of slab gels and cassettes.
  • the inner-most of the two plates, that is the one which is clamped adjacent the support framework, will have a notch which is matched to a similar opening in the upper buffer reservoir, so that when the upper reservoir is filled, electrolyte will spill through the notch and onto the upper surface of the gel.
  • An electrode will be provided in the upper reservoir, so that a charge can be imposed on the upper surface of the gel via the electrolyte.
  • the lower edge of the vertical slab gel is likewise exposed, and when the device is placed in the tank, and the tank partially filled with electrolyte sufficient to contact the lower edge of the gel, the activation of the second electrode provided in the lower reservoir (with opposite polarity to the upper electrode) will cause the establishment of an electrical field with a current flowing through the gel between the upper and lower edges.
  • electrophoretic separation of the sample components is effected as the components are exposed to the electrophoretic separation parameters established in the gel. These parameters can be physical, such as the concentration or density of the gel matrix, or the temperature of the gel matrix, such as when a temperature gradient is established from one region of the gel to the next.
  • Such a temperature gradient will be established using a series of thermal conducting cells, located adjacent to at least one of the glass plates.
  • Each cell will include a source of thermal control, typically a heating element, and a thermal measuring device, typically a thermistor, the combination of which will generally be under the control of a programmable temperature gradient control system, for example a computer controlled system.
  • the temperature control element will be set to operate at a predetermined temperature, and will he maintained at approximately the preset temperature, by the computer control operating on the information provided by the thermal measuring device. This arrangement allows virtually any desired temperature gradient to be established and maintained during the electrophoretic separation.
  • the chemical parameters of the gel can be varied, such as the pH of the gel, or the inclusion of, for example, denaturing agents which will denature certain of the components of the sample.
  • the denaturing agents will be included in the gel in a concentration gradient from one region of the gel to the next.
  • Such gradients will generally be established during the casting of the gel, for example by gradient mixing devices in accordance with means well known in the art.
  • a slab gel will be cast between the two plates, or as a part of a pre-made cassette.
  • the gradient is generally established vertically so that along any horizontal cross section, the gel concentrations will be substantially equal.
  • a sample subject to electrophoretic separation along the first separation path will experience approximately uniform conditions, and the separation will be generally in accordance with the length or molecular weight of the various components.
  • a gel can be cast in a manner that presents a variety of conditions to such a horizontal separation path, without exceeding the scope of the present invention. Disclosure of Specific Embodiments While the present invention is susceptible to a wide range of configurations, arrangements and embodiments, the following discussion will focus on specific examples, the structural and functional aspects of which will serve to provide an understanding of the invention as a whole.
  • FIGS 1 and 2 depict an embodiment of an apparatus 10 in accordance with the present invention, the apparatus including a tank 12 and a lid 14 that fits over the open top of tank 12 to protect the tank contents from external objects and the environment and to reduce evaporation losses from the tank, and whose removal permits easy access to the tank interior.
  • Tank 12 accommodates a core assembly 16 which is designed to accommodate at least one, and preferably at least two, gel cassettes 18 or blotting cassettes 20, as shown in greater detail in, e.g., Figures 2, 8 and 9.
  • an adapter plate typically a piece of Plexiglas
  • the tank 12 is desirably provided as a unified, molded unit, which includes recesses 22, 24 in opposing tank walls 26, 28.
  • Each recess 22, 24 will desirably include a base portion 30, 32 and a retention portion 34, 36 (depicted in Figure 7 as substantially vertical projections).
  • recess 22 is configured to accept, for example, a commercially available leak-resistant plastic ice pack 38 (such as the Freezit® ice pack (Ice-Pak,
  • Tank 12 will also desirably also include an upper rim 40 to provide a more stable mounting surface for lid 14, as well as a region to accommodate the electrode connection features described in greater detail hereafter.
  • tank 12 can also include an optional circular region defined by ridge 42, for the convenience of using a magnetic stir bar (not shown), as is commonly employed in gel electrophoresis to agitate the buffer contained in tank 12 during the electrophoretic separation process.
  • lid 14 includes a set of electrical contacts 44, 46 and cables 48, 50 for connecting the apparatus to a power supply (not shown), 5 and supply voltage to the electrodes, each connection on the lid being readily engageable with a corresponding electrical fitting on one electrode, which is shown in Figure 10 and described below.
  • lid 14 will also include vents 52 to reduce pressure in the interior of the tank chamber during use.
  • Tank 14 will also desirably include at least a pair of holes 54 that are aligned to accept projections 56 on core assembly 16, as depicted in Figures 1 and 2. These paired holes/projections are a useful feature for removing lid 14 from tank 12 after electrophoresis or electroblotting.
  • 'banana plugs' to 5 establish the electrical connection to the apparatus electrodes will increase the mechanical resistance experienced by the user in removing lid 14 from tank 12.
  • users can place their thumbs on projections 56 and curl their fingers under the edge of lid 14, and apply mechanical advantage to lifting lid 14.
  • These paired holes/projections, and/or the lower surface of lid 14, can be configured in a way so as toO prevent incorrect installation, thus insuring that proper polarity is maintained in the system.
  • core assembly 16 comprises a support stand 58 comprising a pair of inner support frames 74, the stand 58 including a plurality of latches 60 and a plurality of cassette backing frames 62, as well as the aforementioned projections 56.
  • Each of the support frame 74 and backing frame 62 will include a sealing gasket 64, 66 to5 enhance the fluid retention properties of the reservoir space 68 that is formed when two cassettes 18 or 20 (or a cassette and the alternative adapter plate) are clamped against support stand 58 by employing cassette backing frames 62 and latches 60.
  • the gel cassettes 18 and/or blotting cassettes 20 are held in a substantially vertical orientation and substantially parallel to each other in contact with support stand 58.
  • O Sealing gaskets 64, 66 serve a number of important functions. These gaskets exert pressure around the perimeter of the gel cassette 18, blotting cassette 20 or alternative adapter plate for at least two reasons: First, they serve to separate the anodal from the cathodal buffer chambers; and second to improve the seal of the plastic gel cassette or plates (so that the gel does not have any contact with surrounding buffer other than through the designated sites at the top and bottom of the gel cassette).
  • the gel cassettes 18 employed in the invention can be conventional in construction, and many designs and constructions are known in the art and widely used.
  • pre-cast gel cassettes such as those manufactured by PAGEgel, Invitiogen, Cambrex, and the like.
  • pre-cast gel cassettes are available in standard sizes and are in wide use due to their convenience and uniformity.
  • manually cast slab gel cassettes are also employed, with no significant alterations needed.
  • Slab gel cassettes generally consist of a pair of rectangular flat plates, preferably of transparent material such as glass or plastic, joined to each other with appropriate spacers to establish a gap of controlled and precisely known width which serves as the gel space.
  • each cassette 18 shown in Figures 2 and 5 appears as a three-layer structure of which the outer two layers are the inert support plates and the inner layer is the gel.
  • the exposed edges of the exemplified gels are the vertical edges (in keeping with the description of a vertical slab gel device), with only one vertical edge 70 of each gel being visible in the view presented. Contacting the exposed edges on each end of cassette 18 are supplies of buffer, either contained in reservoir space 68 or in the bottom of tank 12.
  • FIG. 3 A-D depict the core assembly 16 in an exploded perspective view (3 A), a side elevation (3B), a front elevation (3C) and a downward view of the top edge (3D).
  • the support assembly is shown in Figures 3A-D without cassettes.
  • Each cassette supporting region of core assembly 16 is constructed in three layers, most readily visible in Figure 3 A, an outer backing frame 62, an inner support frame 74 and a set of strips of gasket material 64, 66 between the inner 74 and outer frames 62.
  • the inner frame 74 is directly joined to a paired frame 74 to form the support stand 58, and the outer backing frame 62 is hinge mounted to support stand 58 by cassette hinge pins 98, as best seen in Figures 2 and 3 A.
  • a pair of gel cassettes 18 are inserted in the core 16, fitted into place between inner frames 74 and backing frames 62, and then the latches 60 are pressed over the upper ends of backing frames 62 to form a complete gel core assembly 120.
  • the lower surface of latch 60 will desirably include a slight projection 116 that will cooperate with backing frame recess 118 to provide a more secure clamping force, and a more secure, fluid tight seal between backing frame gasket 66, gel cassette 18, and inner frame gasket 64.
  • a representative pathway for the electrical connection established between the power supply (not shown) and the electrodes 72, 76 can be seen in Figures 1 and 10.
  • cable 48 attaches to contact 44 in lid 14.
  • Contact 44 mates to tank plug 78, which is mounted on rim 40 of tank 12.
  • Tank plug 78 is then connected to core plug 82 by "Z" clip 86, which permits the various alignments of the lid 14, tank 12 and core 16 to be conveniently established.
  • FIGS. 8 and 9 depict a representative blotting cassette 20 as employed in the present system.
  • Blotting cassette 20 is typically constructed from an inert plastic and includes hinged apposing perforated side panels 100, 102, which can conveniently be color-coded (e.g. red and black), so that proper orientation is maintained for the blotting procedure.
  • Each of side panels 100, 102 are fitted with rotatable clamps 104, 106 in order to secure the blotting cassette assembly 124 ( Figure 11) after the various components are assembled prior to electroblotting.
  • blotting cassette(s) 20 can be assembled with the core 16 in a manner analogous to that described previously for gel cassettes 18 to form a complete blotting core assembly (not shown) analogous to complete gel core assembly 120.
  • Cassette side panels 100, 102 are perforated in order to provide a pathway for the electrical gradient established during the electroblotting procedure.
  • the blotting cassette 20 is designed to fits into the cassette support 58 in the same manner as gel cassette 18. To force the electric field solely through the perforations, the perimeter of the blotting cassette is flat and not perforated. This flat and unperforated surface seals against the gaskets 64, 66 embedded in hinged backing frame 62 and support frame 74 faces.
  • a typical blotting cassette assembly 124 will include a foam pad 108, a gel 110 that has previously been used for electrophoretic separation (e.g. in a gel cassette 18), a separation membrane 112 and a sheet of blotting paper 114 positioned adjacent the side of cassette 20 color-coded red (e.g. either panel 100 ox panel 102), all as routinely understood in the art.
  • gel 110 will be oriented so that the higher molecular weight bands contained therein are positioned adjacent to the hinge region of the blotting cassette 20 in blotting assembly 124.
  • This positioning is deemed advantageous as it places the high molecular weight factions closest to electrode 72, and thus enables more power to be applied to the molecular fractions that are slower to migrate in the applied electrical field.
  • the blotting cassette 20 is then positioned in support stand 58 so that the hinge region is lowermost, and the red side is facing toward tank 12 rather than cassette support 58, thus maintaining proper orientation with the electrodes.
  • FIGS. 12 through 14 show details of an alternative embodiment 210 of the present system that includes an active temperature adjustment system, including the provisions for circulation and cooling systems as they appear from the exterior of lid 214 and tank 212.
  • a more conventional tank 212 is utilized, which lacks the tank recesses 22, 24, tank recess bases 30, 32, and tank recess retainers 34, 36, as these are employed in the simplified, passive cooling system described previously. Otherwise, tank 212 will be similar to tank 12. Likewise, lid 214 will include a number of additional features, as described herein below, but will otherwise be similar to lid 14.
  • the active temperature adjustment system circulates a heat exchange medium fluid through access apertures located on the top of the tank through fittings 216, 218 mounted in the lid 214.
  • a conventional pump connected to an external circulation, line draws the fluid solution through the conduit 220 and heat exchange panels 222, 224 and returns it.
  • this heat exchange system will be employed as a cooling system to remove excess heat generated during the electrophoresis separation procedure.
  • the pumps and chilling unit are of conventional design and construction and many such units are available from equipment suppliers. The particular choices of each are not critical to this invention.
  • These heat exchange panels 222, 224 are immersed into the lower buffer tank to maintain the buffer/gel at a desired temperature.
  • the heat exchange panels are typically constructed of acrylic on one side but glass on the other to maximize heat transfer. Desirably, the glass is laminated to the plastic in such a way that internal pressure increases the strength of the seal.
  • These lieat exchange panels 222, 224 also displace an amount of buffer such that less is required for routine electrophoresis.
  • the apparatus of this invention is suitable for use, and readily adaptable if necessary, to slab gel cassettes in general, including cassettes of a wide range of dimensions.
  • the cassettes will be generally square or rectangular, and a typical cassette will have a height of up to about 20cm, and a width likewise up to about 2Ocm, each more commonly approximately 10cm.
  • the gel space between the glass or plastic plates of the cassette will typically be from about 0.5mm to about 3.0mm in width, or preferably from about 0.75mm to ahout 1.5mm, established by appropriate spacers between the plates.
  • the total cassette width, including the plates, will typically be about 1.0 cm or less, more usually approximately 0.8cm or less.
  • the number of cassettes that the cassette supports can accommodate may vary, although two cassettes is generally considered optimal.
  • the tank and accessory parts for the temperature adjustment system (including the tubing and pump) will typically accommodate from about 1 to about 50 liters of buffer (with no cassettes installed in the tank), or most preferably a maximum of about 10 liters. However, when appropriately configured, the present system allows operation with as little as 1 liter or less.
  • the materials of construction are likewise not critical and can vary widely, provided that chemically inert insulating materials are used for the tank, lid, cassette supports, and all other parts.other than the conductive coatings. Clear acrylic or clear polycarbonate are examples of useful materials for the tank and lid, and silicone rubber is an example of a useful material for the gasket strips.
  • the cooling tubes are conveniently made of, e.g., plastic, ceramic or other material that will function effectively as heat exchange tubing.
  • the present apparatus may be operated under conditions that are typical for electrophoretic separations.
  • a typical running voltage may be 2O0 volts dc with a maximum voltage of 1000N, and a typical current per cassette of 30 to 50 mA.
  • a typical buffer solution temperature imposed by the cooling system is 25°C or less, with a minimum of 15°C.
  • the system of the present invention as described herein will permit the rapid and efficient performance of gel electrophoretic separations and analysis, while decreasing both the amount and the cost of the labor associated with such assays, and will facilitate standardization of such assays as well.
  • the improvements due to the use of the present system, particularly in combination with PCR robots and fluorescent image analysis, should lead to further efficiencies, accuracies, and cost reductions.
  • the present system operates with all protocols ordinarily employed in, for example, SDS-PACrE, electroblotting (e.g. Western blotting), acrylamide nucleic acid separations and the like.
  • electroblotting e.g. Western blotting
  • acrylamide nucleic acid separations e.g., acrylamide nucleic acid separations and the like.
  • Representative examples of the assembly and use of a specific embodiment of the present system in such protocols are described below, for purposes of illustration rather than limitation.
  • a plastic adaptor plate is fitted into the side without the gel cassette. If a Cambrex 10cm x 9cm gel cassette is to be used, then a shim will be required. 6. Close the backing frame(s) and relatch them to the central support stand by pressing down on the latches. 7. -A stirring bar can be placed into the bottom of the reservoir tank in the stirring corral region. 8. If using freezer blocks to provide cooling, the frozen blocks are removed from the freezer and inserted into the recess receptacles on either side of the reservoir tank.
  • the core assembly is placed into the reservoir tank.
  • the anode (red) and cathode (black) electrodes are color-coded on both the core/cassette assembly and the reservoir tank.
  • the user should ensure that the red dot on the cassette assembly is on the same side as the red receptacle on the reservoir tank.
  • the upper core reservoir formed by the insertion of the gel cassette(s) or adapter plate, is filled with freshly prepared buffer ( ⁇ 190mL). If any buffer is spilled into the plug jack receptacles in the reservoir tank, these should he dried completely using compressed air. Failure to do this will result in accelerated plug jack corrosion. 2. Important note if using the optional freezer cooling blocks: Each freezer block displaces approximately 125mL of buffer.
  • Gel Casting Using Gel WrapTM Gasket Casting Method 1. Required components: glass plate set, Gel " WrapTM gasket, spacer set, comb, three GPC-0002 clamps, and polyacrylamide solution. Prepare and clean glass plates by hand washing both plates with a high quality lab detergent following by a complete rinsing with dH 2 O. Air-dry or use a lint-free tissue. Spray/wipe the chosen inner surfaces of the plate set with 95% ethanol and dry with lint-free tissue. 2. Start gel casting procedure by holding the 3mm thick, notched back plate with the rounded bottom corners and applying the gasket around one side of the glass plate.
  • one side of the "U" shaped gasket is flat, and the other side has tubing that will act as a seal around the spacers.
  • Preparing the Unit for Blotting 1. Remove safety cover lid from the assembled unit by simultaneously pressing down on projections while lifting up on lid. Remove core from reservoir tank by grasping core with one hand and lifting directly up. Open the core assembly by pulling up on the latches. 2. Open blotting cassette and lay it flat on the bench. 3. Assemble blotting stack as shown in Figure 11. With cassette wide open assemble components on black side in the following order: foam pad, gel*, buffer saturated transfer membrane, then buffer saturated blotting paper. Smooth with glove finer or roll with glass rod to be sure no bubbles exist between the gel and the transfer membrane.
  • Electro-Blotting Procedure Place stirring bar in bottom corral of lower reservoir tank. Place frozen freezer blocks in side receptacle recesses. Place core/blotting cassette assembly into lower reservoir. The anode (red) and cathode (black) electrodes are color-coded on both the core/cassette assembly and lower reservoir. Ensure the red dot on the cassette assembly is on the same side as the red receptacle on the lower reservoir. 2. Pour 1 liter of freshly prepared, chilled (4°) buffer into lower buffer reservoir. Buffer will percolate into central core. 3. Attach safety cover lid. The unit is ready for power. 4. Connect the leads to the power supply, matching the color-coded red to red and black to black. Begin transfer by electrophoresis. Removing the Blot 1.
  • Blotting cassettes can be removed by leaving the core in place and opening the top latches of the core, opening the backing frames and lifting the cassettes out. Unlatch the blotting cassettes and remove blot from blotting sandwich.

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Abstract

La présente invention a trait à un appareil pour l'électrophorèse en gel permettant la réalisation par l'utilisateur d'une séparation électrophorétique et un électrobuvardage dans le même système. L'invention a également trait à des moyens d'échange thermique avec le système, par exemple pour le refroidissement du système lors de son fonctionnement.
PCT/US2005/011091 2004-04-01 2005-04-01 Electrophorese en gel multifonctionnelle et appareil WO2005098408A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102053113A (zh) * 2009-11-06 2011-05-11 张利群 一种带有冷却腔的电泳槽
WO2011072158A1 (fr) * 2009-12-11 2011-06-16 Bio-Rad Laboratories, Inc. Instrument d'électrotransfert indépendant dans des cassettes multiples
KR20120112531A (ko) * 2009-12-10 2012-10-11 바이오 래드 래버러토리스 인코오포레이티드 일체형 전기 접점과 잠금 메카니즘을 갖는 전기 전달 카세트
EP2678671A1 (fr) * 2011-02-24 2014-01-01 Bio-Rad Laboratories, Inc. Stabilisation dimensionnelle de cassettes de gel en plaques pour empêcher une distorsion provoquée par le gonflement des gels
WO2018228447A1 (fr) 2017-06-13 2018-12-20 Nanjingjinsirui Science & Technology Biology Corp. Dispositif de transfert rapide et ses applications
WO2020079211A1 (fr) * 2018-10-18 2020-04-23 Danmarks Tekniske Universitet Ensemble d'électrophorèse
US20220011264A1 (en) * 2020-07-13 2022-01-13 Life Technologies Corporation Electrophoresis & electrotransfer devices, systems, & methods
WO2022195009A3 (fr) * 2021-03-19 2022-10-27 Global Life Sciences Solutions Operations UK Ltd Cassette de transfert sur membrane
USD975873S1 (en) 2020-07-13 2023-01-17 Life Technologies Corporation Electrophoresis and electrotransfer device
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EP2509704A4 (fr) * 2009-12-10 2014-09-03 Bio Rad Laboratories Cassette d'électrotransfert dotée de contacts électriques intégrés et d'un mécanisme de verrouillage
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WO2011072158A1 (fr) * 2009-12-11 2011-06-16 Bio-Rad Laboratories, Inc. Instrument d'électrotransfert indépendant dans des cassettes multiples
CN102711904A (zh) * 2009-12-11 2012-10-03 美国伯乐公司 在多个盒子中独立电转移的装置
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CN110753839B (zh) * 2017-06-13 2022-09-09 南京金斯瑞生物科技有限公司 快速印迹装置及其应用
CN110753839A (zh) * 2017-06-13 2020-02-04 南京金斯瑞生物科技有限公司 快速印迹装置及其应用
EP3639017A4 (fr) * 2017-06-13 2021-03-17 Nanjing GenScript Biotech Co., Ltd. Dispositif de transfert rapide et ses applications
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WO2018228447A1 (fr) 2017-06-13 2018-12-20 Nanjingjinsirui Science & Technology Biology Corp. Dispositif de transfert rapide et ses applications
WO2020079211A1 (fr) * 2018-10-18 2020-04-23 Danmarks Tekniske Universitet Ensemble d'électrophorèse
US20220011264A1 (en) * 2020-07-13 2022-01-13 Life Technologies Corporation Electrophoresis & electrotransfer devices, systems, & methods
WO2022015661A3 (fr) * 2020-07-13 2022-04-14 Life Technologies Corporation Dispositifs, systèmes et procédés d'électrophorèse et d'électrotransfert
USD975873S1 (en) 2020-07-13 2023-01-17 Life Technologies Corporation Electrophoresis and electrotransfer device
WO2022195009A3 (fr) * 2021-03-19 2022-10-27 Global Life Sciences Solutions Operations UK Ltd Cassette de transfert sur membrane
WO2023164488A1 (fr) * 2022-02-25 2023-08-31 Life Technologies Corporation Système et procédés de transfert de biomolécules

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