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
• • 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
• • 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
  • G01N27/44778—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis on a common gel carrier, i.e. 2D gel electrophoresis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
  • G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T292/00—Closure fasteners
  • Y10T292/08—Bolts
  • Y10T292/0801—Multiple
  • Y10T292/0834—Sliding
  • Y10T292/0836—Operating means
  • Y10T292/0837—Cam and lever

Definitions

• This invention resides in the field of gel electrophoresis, and relates in particular to the transfer of electrophoretically separated species from a slab gel in which the species were separated to a sheet-form support matrix in which the species can be detected, identified, and quantified.
• Proteins, nucleic acids, or other biological species that have been electrophoretically separated in a slab gel are often transferred to a membrane of nitrocellulose, nylon, polyvinyl difluoride, or similar materials for identification and quantification which are more easily performed on the membrane than in the gel.
• a common transfer technique is electrob lotting, in which the flat surfaces of the gel and membrane are placed in direct contact and an electric current is passed through both the gel and the membrane in a transverse direction, thereby transferring the species in a manner similar to that by which the species were mobilized within the gel.
• the transfer is termed a Southern blot after its originator, the British biologist Edwin M. Southern.
• RNA fragments is termed Northern blotting
• Western blotting the transfer of proteins or polypeptides is termed Western blotting.
• the species on the membrane are analyzed by methods appropriate to the species themselves.
• Southern and Northern blots for example, the analysis involves treatment of the species on the membrane with a hybridization probe, followed by labeling them with a fluorescent or chromogenic dye.
• Western blots the species are treated with antibodies, followed by the use of conventional labeling techniques to detect the antibodies.
• Electroblotting of the Southern, Northern, and Western types can all be performed in either a wet, dry, or semi-dry format.
• wet blotting the gel and membrane are layered over each other in a stack which is immersed in a transfer buffer solution in a tank on whose walls are mounted wire or plate electrodes. The electrodes are then energized to cause the solutes to migrate from the gel to the membrane.
• semi-dry blotting filter papers wetted with the transfer buffer solution are used, and the stack contains the filter papers on the top and bottom with the gel and the membrane between the filter papers to form a "blotting sandwich.” The electrodes are then placed in direct contact with the exposed surfaces of the wetted filter papers. Dry electroblotting uses no liquid buffers other than those residing in the gels. Descriptions of wet, dry, and semi-dry electroblotting and the associated materials and equipment are found in Margalit et al. (Invitrogen) United States Patent Application
• the present invention resides in an electrotransfer cassette of unique construction.
• electrotransfer cassette is used herein to mean any receptacle that contains electrodes and can accommodate a gel or other medium that has chemical or biological species distributed therein in a two-dimensional array such as the wells of a microtiter plate, plus a membrane or other two-dimensional matrix to which the species are to be transferred by the influence of the electric field generated by the electrodes. While broad in application, the cassettes of this invention are of particular value for use in semi-dry electroblotting.
• the cassette construction includes a top part and a bottom part, with the two electrodes in the form of flat plates mounted on the top and bottom parts, respectively.
• the anode is preferably in the bottom part and the cathode in the top part, although the reverse
• cathode support In all locations where the term "cathode support” is used, it is understood that the cathode plate in that support can be replaced by an anode plate, and vice versa for the term "anode support.”
• the cathode and anode supports are readily secured together and readily removed from each other by a locking and releasing mechanism that is manually operable from the cathode support.
• the mechanism includes pegs that extend laterally from opposing side edges of the cathode support and that are aligned with apertures in raised walls of the anode support.
• the pegs are movable between extended positions where the pegs protrude through the corresponding apertures and retracted positions where the pegs are withdrawn into the cathode support and out of engagement with the apertures.
• the former is a locking position in which the cathode and anode supports are secured together by way of the extended pegs and the engagement of the extended pegs by the apertures.
• the latter is a release position in which the cathode and anode supports can be separated from each other, or placed in contact prior to being locked in place by the pegs. Movement of the pegs is achieved by a finger grip that can be manipulated by a single hand of the user and that shifts the positions of all pegs simultaneously between the two positions.
• the finger grip is a rotary disk in the cathode support that is joined to the pegs through any conventional mechanical connection that translates rotary motion to linear motion.
• An example is a rack-and-pinion gear, where a toothed circular gear fixed to the rotary disk engages toothed bars on two sides, where each bar is an extension of a frame or web to which the pegs are mounted.
• Rotation of the circular gear causes the bars and hence the frames and pegs to move simultaneously in opposite directions, i.e., outward for the pegs to protrude through the apertures or inward to withdraw from the apertures, depending on the direction of rotation of the circular gear.
• the locations of the apertures in the raised walls of the anode support of the cassette fix the height of the cathode support above the anode support, and hence the distance between the cathode and the anode.
• the pegs and apertures also hold the electrodes parallel to each other. Alternatives to the rotary disk will be readily apparent to those skilled in the art.
• cassettes in preferred embodiments of the invention include a two-portion cathode (i.e., upper electrode) support in which the electrode plate is affixed to a lower portion and the rack-and-pinion mechanism is mounted to an upper portion.
• the upper and lower portions are joined by mating members in a snap-on connection of variable but spring-loaded separation to allow the electrode plate to be raised or lowered to accommodate blotting sandwiches of different thicknesses.
• the cassette contains anodic and cathodic electrical contacts that are accessible from the exterior of the cassette for electrical connection to a power supply.
• the electrical connections to these contacts are made by simple contact, such as upon insertion of the cassette into an instrument with internal power connections.
• Still further features in preferred embodiments of the invention are integrated gasket features that isolate, or maintain a separation between, the interior surfaces of both the top and bottom portions of the cassette, on one hand, and the gel and membrane or other blotting materials, on the other. These gasket features preserve the structural materials of the cassette from the chemical deterioration that might otherwise result from contact with the chemicals in the gel and the buffers. Still further features will be apparent from the descriptions that follow and the accompanying figures.
• FIG. 1 is a perspective view of an example of a cassette in accordance with the present invention.
• FIGS. 2 A and 2B are top and bottom views, respectively, of the cathode support of the cassette of FIG. 1, showing the movable pegs.
• FIGS. 3 A and 3B are top and bottom views, respectively, of the anode support of the cassette of FIG. 1.
• FIG. 4 is a perspective view of the cassette of FIG. 1 from the rear.
• FIG. 5 is a plan view of the rack-and-pinion mechanism for locking and releasing the cassette of FIG. 1.
• FIGS. 6A and 6B are views of the two positions, respectively, of the rack-and- pinion mechanism of the cassette of FIG. 1.
• FIG. 7 is an exploded view of an alternative cathode support of a cassette within the scope of this invention, the cathode support itself being formed in two portions.
• FIG. 8 is a cross section of a segment of a cassette using the alternative cathode support of FIG. 7.
• FIG. 9 is a view of the same cross section as FIG. 8, expanded to accommodate a thicker blotting sandwich.
• FIG. 1 depicts one example of an assembled cassette 11 within the scope of this invention, while the separated cathode support 12 and anode support 13 are depicted in the succeeding Figures.
• the cathode support 12 is shown from above in FIG. 2 A and from below in FIG. 2B.
• the anode support 13 is shown from above in FIG. 3 A and from below in FIG. 3B. Both supports are rectangular in shape; the anode support 13 has raised walls 14, 15, 16, 17 on all four sides to receive the cathode support 12 in a close but loose fit so that the cathode support 12 can easily be inserted inside these walls and removed.
• the cathode and anode themselves are visible in FIGS. 2B and 3A, respectively, the anode 21 residing on the floor of the anode support (FIG. 3 A) and the cathode 22 on the underside of the cathode support (FIG. 2B).
• the anode support 13 has an integrated gasket 23 (FIG. 3 A), which can be overmolded onto the floor of the support.
• the gasket extends around the periphery of the floor of the anode support and encircles the anode 21.
• the blotting sandwich is placed on the anode inside the gasket 23.
• the cathode 22 (FIG. 2B) contacts the upper surface of the blotting sandwich.
• a raised ridge 24 (FIG. 2B) is formed on the underside of the cathode support. The raised ridge 24 can be formed of additional gasket material overmolded onto the cathode support.
• the gasket 23 and the raised ridge 24 prevent the chemicals in the blotting sandwich from contacting parts of the cassette other than the anode and cathode. Gases generated during the blotting proceess can pass from the cassette through the gap between the gasket 23 and the raised ridge 24 to escape the cassette through the gaps between the long edges of the cathode support and the long side walls of the anode support.
• FIG. 3B Further features of the cathode and anode supports are overmolded feet 25 on the underside of the anode support (FIG. 3B), and similarly overmolded corner pads 26 on the four corners of the upper surface of the cathode support (FIG. 2A).
• the feet 25 are useful in stabilizing the cassette on a laboratory bench.
• the corner pads 26 help center the cathode support in the anode support and facilitate the insertion of the cassette into the instrument.
• the movable pegs 31 (FIG. 2A) are part of the cathode support 12, which contains two pairs of such pegs, one pair on each of the two longer side edges of the top part, extending outward.
• the apertures 32 (FIG.
• the rotary disk 33 that governs the positions of the pegs is integrated into the cathode support 12 (FIG. 2 A), and discussed in more detail below.
• the pegs are preferably oriented with their axes parallel to the cathode plate, and their motion when shifting between the locking and release positions is along these axes.
• the cathode and anode supports are both rectangular in outline, each having a pair of opposing parallel side edges on their longest sides.
• the locations of the pegs are not critical provided that they maintain the anode and cathode plates in a parallel relation when securing the two supports together.
• at least one peg resides on each of the two parallel side edges, more preferably two or more on each side, as shown.
• External contacts for the cathode and anode plates reside in the cathode and anode supports of the cassette, respectively, in one end wall of each support.
• a flat area on the outer surface of the back end wall 15 serves as a single anode contact 34.
• two protrusions 35, 36 with flat ends extending outward from the back edge of the cathode support serve as cathode contacts.
• the contacts are extensions of the anode and cathode plates, respectively, bent to a vertical position.
• the anode and cathode can be reversed, with the anode can be in the top part of the cassette and the cathode in the bottom part and the external contacts reversed correspondingly.
• control of the height of the cathode above the anode and the parallel spacing between them is achieved by features on the floor of the anode support adjacent to the front end wall 17 (FIG. 3 A, although the features are not visible in the drawing) and by heights of the bottoms of the slots 37, 38 in the back end wall 15 as the cathode support 12 rests on these features and slots.
• FIG. 5 The rack-and-pinion mechanism governing the positions of the pegs 31 is shown in FIG. 5.
• the pegs are extremities of frames, the four pegs thus being part of two frames 41, 42.
• At the inner end of each frame is a toothed bar 43, 44, the two sets of teeth (one set on each bar) engaging opposite sides of a circular gear 45.
• Rotation of the circular gear in one direction thus causes both frames to move outward, and hence both pairs of pegs to protrude further outward, and rotation in the other direction causes both frames, and hence both pairs of pegs, to move inward.
• the circular gear 45 is affixed to the underside of the rotary disk 33.
• FIGS. 6A and 6B are horizontal cross sections of the cathode support, showing the action of the rotary disk and moving the pegs. The locking position is shown in FIG. 6A and the release position is shown in FIG. 6B.
• FIG. 7 depicts the cathode support 51 of the cassette, the cathode support itself being formed in two portions, a shell 52 and an electrode plate mount 53 with the shell fitting over the mount. A cathode plate (not visible) is secured to the underside of the electrode plate mount 53
• a rack-and- pinion mechanism although not shown in FIG. 7, is secured to the underside of the shell 52 at the location of the circle 54, and has the same structure as that shown in FIG. 5.
• the movable pegs 31 (FIG. 5) that extend from the rack-and-pinion mechanism pass through lateral apertures 55 in the shell to engage the lateral apertures 32 (FIG. 3A) in the bottom part of the cassette.
• each mating pair consisting of a hollow boss or shell 56 extending downward from the upper portion 52 and a set of resihently mounted hooks 57 extending upward from the lower portion 53.
• Each set of hooks consists of four hooks with arcuate cross sections, arranged in a circle.
• Each hollow boss 56
• each hollow boss 56 terminates in a widened rim 58 or inverse flange (with a lip extending toward the center axis of the boss), and the hooks 57 face outward.
• the hollow bosses 56 are aligned with the hooks 57 and when the parts are joined, each hollow boss 56 fits over a corresponding set of four hooks 57 and all four hooks snap into place to engage the rim 58 of the boss.
• Spring- loaded biasing members can be included to contact both the shell and the electrode plate mount to urge the plate mount downward from the shell and yet to allow the plate mount to be pushed upward upon contact with the blotting sandwich to accommodate a relatively thick blotting sandwich between the two electrode plates.
• the spring-loaded biasing members are represented by a coil spring 59 inside each boss 56.
• FIG. 7 Also shown in FIG. 7 are electrical contacts 61, 62, protruding from opposing edges of the upper portion 52 corresponding to the cathode contacts 35, 36 in the structure shown in FIG. 4.
• the placement of the contacts 61, 62 on opposing edges allows the cassette to be inserted into an instrument in either direction.
• the movable pegs protruding laterally from the cathode support of the cassette in the FIG. 7 embodiment are at a fixed height relative to the upper portion 52 of the cathode support and the apertures 32 are at a fixed height in the anode support (FIG. 3 A)
• the lower portion 53 of the cathode support i.e., the portion to which the cathode plate is mounted
• the position of the lower portion 53, and hence the height of the cathode plate determine the vertical distance between the cathode plate and the anode plate.
• the height self-adjusts to accommodate the thickness of the blotting sandwich.
• the user When assembling the cassette over a blotting sandwich, therefore, the user places the blotting sandwich over the anode on the bottom part of the cassette, then places the top part of the cassette over the blotting sandwich and presses down on the top part, compressing the springs, until the pegs are aligned with the apertures in the bottom part.
• the pressure on the cathode plate from below causes compression of the springs 59, while for a thin blotting sandwich, the springs are relatively relaxed.
• FIGS. 8 and 9 Two conditions of the springs and thus two heights of the cathode plate are shown in FIGS. 8 and 9, respectively.
• Both Figures are cross sections of a portion of the assembled cassette with a blotting sandwich between the anode and cathode plates, the portion shown being that which is in the immediate vicinity of one of the pairs of mating members joining the two portions of the cathode support.
• the parts shown include a hollow boss 56 with a inwardly-extending rim 58, three of the set of four hooks 57 (two in cross section), the coil spring 59, the cathode plate 22, and the anode support 13 which includes an anode plate 21.
• the blotting sandwich (also not shown) has been placed between the cathode and anode supports and fills the gap 63 between the cathode plate 22 and the anode plate 21, setting the height of the gap as the spring 59 urges the cathode plate against the top of the blotting sandwich.
• a thicker blotting sandwich is placed between the cathode and anode supports so that when the movable pegs in the cathode support are aligned with the apertures in the anode support to secure the cassette together, the blotting sandwich presses the cathode plate 22 upward against the springs 59.
• the spring 59 is compressed and the gap 63 is wide enough to accommodate the thicker blotting sandwich.

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• 2010
  • 2010-12-08 US US12/963,417 patent/US8357278B2/en active Active
  • 2010-12-09 CA CA2782021A patent/CA2782021C/en not_active Expired - Fee Related
  • 2010-12-09 JP JP2012543292A patent/JP5449568B2/ja not_active Expired - Fee Related
  • 2010-12-09 WO PCT/US2010/059770 patent/WO2011072167A1/en active Application Filing
  • 2010-12-09 EP EP20100836715 patent/EP2509704B1/en active Active
  • 2010-12-09 CN CN201080055897.8A patent/CN102725052B/zh active Active
  • 2010-12-09 KR KR1020127017879A patent/KR101721718B1/ko active IP Right Grant
• 2012
  • 2012-05-24 IL IL219985A patent/IL219985A/en active IP Right Grant

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