US3719580A - Electrophoretic apparatus - Google Patents

Abstract

An electrophoretic apparatus in which the gel medium is set while disposed in a vertical attitude, the apparatus including vertically arranged first and second buffer solution containers defining therebetween a zone or cavity for holding a gel-forming solution, the latter solidifying in situ to form an electrophoresis gel medium. The apparatus is further characterized in that it includes a fluid flow barrier which separates the buffer containing chamber from the gel medium cavity, a preferred form of the barrier constituting a semipermeable membrane which permits the passage of electrically charged particles therethrough but precludes gross transfer of liquid between the buffer chamber and the gel-containing cavity.

Description

United States Patent Roberts et a].

[ 1 March 6, 1973 Primary ExaminerT. Tung Assistant Examiner-A. C. Prescott Attorney1(egan, Kegan & Berkman [5 7] ABSTRACT An electrophoretic apparatus in which the gel medium is set while disposed in a vertical attitude, the apparatus including vertically arranged first and second buffer solution containers defining therebetween a zone or cavity for holding a gel-forming solution, the latter solidifying in situ to form an electrophoresis gel medium. The apparatus is further characterized in that it includes a fluid flow barrier which separates the buffer containing chamber from the gel medium cavity, a preferred form of the barrier constituting a semipermeable membrane which permits the passage of electrically charged particles therethrough but precludes gross transfer of liquid between the buffer chamber and the gel-containing cavity.

18 Claims, 10 Drawing Figures PATENTEOMR SHEET 10F 2 I /0 INVENTORS.

JOHN s. JONES RICHARD M. ROBER T5 ELECTROPHORETIC APPARATUS This invention relates to improved electrophoretic apparatus and techniques for effecting the electrophoretic separation of electrically charged molecules traversing an electric field. More particularly, the invention is directed to an electrophoretic apparatus in which the gel-forming medium is introduced into the apparatus while the gel-defining cavity is disposed in a vertical position and in which the intermixing of the gel-forming solution with buffer solution contained in a buffer retaining chamber is obviated.

It is a principal object of the invention to provide an improved electrophoretic apparatus which avoids the shortcomings and the objectionable features of prior art devices and which provides, in a simple and highly functional structure, advantages not heretofore realized.

Another object of the invention is to provide an electrophoretic apparatus in which the gel pockets into which the samples to be analyzed are introduced are readily and directly accessible from above.

A related object of the invention is to provide an improved electrophoretic apparatus in which a plurality of sample receiving gel pockets may be loaded simultaneously and in a most simple manner.

It is an important feature of the apparatus of the invention that there is no necessity to pour the buffer solution from the buffer retaining chambers in order to remove the gel.

Still another feature of the invention is that it provides a simple technique through which a gel may be cast vertically so as to facilitate the use of methods invoking the principles associated with and the advantages of density or concentrational gradients, a practice not possible with horizontally cast gels.

A related feature of the invention is that it provides an important technique and apparatus which eliminates any wasting of the buffer solution and which obviates any need for premature discard of the solution.

Yet another feature of the apparatus of the invention is that the buffer solution can be quickly and easily poured from each of the buffer containing chambers, either individually or simultaneously.

It is a general object of the invention to provide an improved electrophoretic apparatus which is simple in design, inexpensive to fabricate, and convenient to use.

It is an important feature of the improved apparatus of the invention that it eliminates the necessity for casting the gel material while the latter is disposed in a generally horizontal plane, and provides a unique technique and apparatus in which the shifting of the apparatus from an initial horizontal disposition to an ultimate vertical disposition for carrying out vertical gel electrophoresis is obviated.

Another important feature of the apparatus of the invention is that it facilitates the use of any preferred, selectable gel thickness, all achieved without any major modifications of the equipment involved.

Electrophoresis has been undeniably established as one of the most powerful scientific research tools and techniques developed in recent years for the separation of closely related molecules. Generally, the technique relies on differences in net charges to effect separation of materials physically, by exposure to an electric field. In this field, molecules of greatest net charge migrate at the fastest rate toward the pole of opposite charge, outdistancing those molecules of lesser net charge and thus producing a physical or spatial separation. The overall effect is to separate, simultaneously, the different species of molecules from each other. While early electrophoretic techniques utilized a liquid medium, later workers have found that the use of a solid or a gel as a supporting medium for the substances under investigation constitutes a superior method. It has also been demonstrated that certain gel media contribute to the effective separation of the molecules by exerting a sieving effect, whereby the size and shape of the molecule as well as the charge carried as the molecules migrate toward the poles of the electric field, contribute to effective and definitive separation.

The first non-liquid supporting media to be used in electrophoretic techniques were filter paper and hydrolyzed plant starch. These materials are objectionable because of the poor separation achieved and because of difficulty in handing the materials or the excessive time required to effect the separation. Many other supporting media have been investigated and tested, and the medium most widely used at the present time is synthetic polyacrylamide gel. Some of the advantages of this medium over other materials are its ease of use, its high strength and flexibility, its transparency, and its ease of storage. Most important, however, is that the pore size, responsible for the sieving effect, and the charge of the gel itself can easily be controlled, making acrylamide gel suitable for the electrophoretic separation of an extremely wide variety of substances.

Electrophoresis is now a technique used in many fields of biology and medicine. In biochemistry, it has become a standard technique for the purification of such substances as enzymes, protein, histones, nucleic acids, and other macromolecules. Of particular importance is the discovery that enzymes of identical activity can actually exhibit different electrophoretic mobility'. If these enzymes are controlled by different genes, they are called isozymes. If they are controlled by different forms of the same genes (and are, therefore, the product of alleles) they are called allozymes. In embryology, electrophoresis has shown that the proportion of different isozymes alters with time and tissue type during embryonic development. Investigation of these differences may provide valuable information about the nature of differentiation.

In genetics, one of the most interesting findings to arise from gel electrophoresis has been the discovery of enormous amounts of genetic variability in enzymes in natural populations. The mechanisms whereby this variability is maintained are still very much a matter of controversy, and many workers are at present investigating this problem. The study of isozymes has also become extremely important to students of evolution, because taxonomic relationships can be worked out by comparing the proportion of genes which different species bear in common. In medicine, electrophoretic separation of hemoglobins and serum proteins is now an important aid in the diagnosis of many diseases because diseased or malfunctioning tissues may fail to produce certain isozymes, or produce different ones. It is likely that electrophoresis will become a standard part of the hospital physicians array of techniques.

Acrylamide gel electrophoresis is now ordinarily carried out in commercia gel boxes which are not only expensive and cumbersome, but also have objectionable features and short-comings. In a typical such apparatus the liquid gel material is poured between two cooling plates while the apparatus is disposed in a horizontal position. During this pouring and gelling period, the gel space is plugged or blocked with a cellulose sponge. A simple pocket former, placed in the top end of the assembly, is cut out and carefully removed just after polymerization of the gel leaving pockets at the upper end of the gel. With the box shifted to a vertical position, buffer solution is introduced into the two chambers. Thereafter, the sample or samples are placed in the pockets using a micro-syringe. For the particular apparatus described, the procedure involved is extremely difficult calling for meticulous care and requiring a high degree of skill. Since the pockets are narrow and the needle of the micro syringe must extend into the pocket at an angle from the top, and since the gel is transparent, the needle may easily pierce the gel by mistake. In such cases the sample is lost when the syringe plunger is depressed. It is only after considerable time that a technician will ordinarily develop sufficient skill to hit the pocket nearly each time. The sample is usually mixed with a percent sucrose solution which, being heavier then the buffer, will cause the sample solution to fall to the button of the pocket and define a substantially even layer.

Using the techniques described, a skilled technician can prepare and load four gel boxes (or 48 samples) in approximately 2 hours. The gels are run for l to 3 hours, depending on the particular assay. The current is then switched off, the buffer poured out of the chambers, and the apparatus disassembled, allowing removal of the gel.

It is to the elimination of the above-indicated difficulties with prior art apparatus and to the simplification of the techniques of electrophoretic analysis that the subject invention is directed. Other and further objects and advantages of the invention will become apparent from the reading of the following specification taken in conjunction with the drawings in which:

FIG. 1 is a perspective view of the electrophoretic apparatus of the invention, showing the cooperating components thereof;

FIG. 2 is a fragmentary perspective view showing one means of securement of the two principal containers of the apparatus to each other;

FIG. 3 is an exploded perspective view showing the two principal containers of the apparatus and the spacer element to be interposed therebetween to establish the gel receiving cavity;

FIG. 4 is a cross-sectional view taken on the line 4- 4 of FIG. 1;

FIG. 5 is a view taken substantially on the line 5-5 of FIG. 3 and showing one mode of securement of the fluid barrier in the slot formed in a wall of a principal buffer containing vessel and showing the passages through which cooling fluid is circulated in the vessel wall;

FIG. 6 is a view taken substantially on the line 66 of FIG. 3 and showing the cooling passages in a principal wall of the second buffer-containing vessel;

FIG. 7 is a cross-sectional view taken on the line 7- 7 of FIG. 3;

FIG. 8 is a cross-sectional view taken on the line 8- 8 of FIG. 3;

FIG. 9 is an enlarged, fragmentary, cross-sectional view taken 'on the line 99 of FIG. 5 and showing one preferred mode of securing the fluid barrier in the wall of the buffer-containing vessel; and

FIG. 10 is a perspective view of a preferred comblike pocket former utilized in conjunction with the electrophoresis apparatus of the invention.

The objects of the invention are achieved and the advantages are realized by providing a vertical gel electrophoretic apparatus which includes, in combination, a pair of opposed buffer-solution-containing vessels spaced apart from one another and defining therebetween a cavity for an electrophoresis gel medium. In a preferred embodiment of the invention one path of ion flow communication between the buffer solution and the electrophoresis gel is achieved through a semi-permeable membrane secured in a window formed in a wall of one of the buffer carrying containers. The spaced vessel assembly is secured by means of clamps to establish a structural unit in which the gel-forming medium is conveniently poured to produce a sheet of vertically extending gel, the technique and structure utilized precluding an intermixing or deffusing of the gel-forming medium into the buffer solution and also preventing contamination of the gel-forming medium by the buffer solution.

Referring now to the drawings, and particularly to FIGS. 1 through 3, the electrophoretic assembly or apparatus 20 is shown, for purposes of disclosures, as embodying, in a preferred embodiment, a first vessel'or container 24 for retaining buffer solution 26, a second 'vessel or container 28 for buffer solution, and a generally U-shaped spacer 30. The spacer 30 is positioned between facing inner walls 34 and 38 of the respective first and second buffer containers 24 and 28 to define therebetween an upwardly opening downwardly extending cavity 40, the cavity being adapted to receive therewithin a fluid gel-forming solution which is solidifiable or polymerizable in situ to form an electrophoresis gel medium 44. Spring-tensioned clamping means 50 are fastened to bridge end walls 54 and 56 and end walls 58 and 60 of the buffer containers 24 and 28 to urge the containers resiliently toward one another and to secure the containers against the interposed spacer 30 in fluid sealing engagement therewith.

As seen most clearly in the exploded view of FIG. 3, the buffer containers or chambers 24 and 28 are formed on facing inside walls or panels 34 and 38 with marginal grooves 64 and 68 which bound or frame the periphery of the panels at the base portion and side edge portions thereof. Seated within and preferably frictionally secured in the grooves 64 and 68 are gaskets, bands, or beads 72 and 76 which, in the preferred embodiment of the invention illustrated, constitute flexible and pressure responsive resilient plastic tubing a continuous and uninterrupted portion of which extends slightly outwardly of the respective faces of the panels 34 and 38 to provide an effective fluidtight sealing contact with the U-shaped spacer 30 which is interposed between the two buffer retaining containers or chambers 24 and 28. It is contemplated that any other sealing expedient among many which are known in the relevant art may be used to effect the required fluid seal, and all such equivalent techniques are considered to be within the scope of the invention as conceived and contemplated.

As indicated diagrammatically in FIG. 3 and as shown more clearly in FIGS. 4, 5, and 6, the walls or panels 34 and 38 which are presented against and abut the U-shaped spacer 30 are formed with internal channels 80 through which cooling (or heating) fluid may be circulated so that heat generated through the use of the apparatus may be effectively removed or dissipated. Suitable input 84 and exhaust ports 88 which extend through the lateral marginal wall portions of the panels 34 and 38 communicate with the exterior of the panels. Any suitable connectors or fixtures (not shown) may be attached to the panel ends at these ports to provide the necessary means for connection of fluid input leads and exhaust lines. In the preferred embodiment of the invention illustrated, the establishment of the fluid channels in the panels 34 and 38 is conveniently achieved by constructing each of these panels from two separate sheets of plastic. Using this expedient, the face of one of the panels is machined or milled to provide open grooves or furrows 80 (FIGS. 5 and 6). The second sheet is then sealed against the first to provide the required fluid passageways or channels.

It will be appreciated from the foregoing discussion that each of the containers 24 and 28 constitutes a buffer solution retainer and that the cavity defined between the facing walls or panels 34 and 38 of the containers, and whose width is dictated by the thickness of the spacer 30, constitutes a zone in which the gel-forming solution is introduced to solidify in situ. Extending across the lateral expanse of the container 24 and secured in the opposed end walls 54 and 58 thereof is an electrode 92 which takes the form of a fine taut wire. A lead line 94 connected to the electrode 92 is fastened to a terminal or port 98 secured on the upper face of the end walls 54, as indicated in FIG. 1. A similar electrode 102 extends across the container 28 between opposed end walls 56 and 60. A previously described, a conductive lead line 104 interconnects the electrode 102 with a port or terminal 108 secured to the top of the end wall 56. Suitable electrical leads connect the terminals 98 and 108 to a power source (not shown), the latter supplying the required EMF for carryin g out the electrophoresis operation.

Referring now to FIGS. 3 through 5 and FIG. 9, a most important feature of the invention constitutes a unique structure and technique through which the passage of liquid between the buffer containing chamber 24 to the gel-containing cavity 40 is precluded while permitting the passage of charged particles. As indicated in FIG. 4 and as seen most clearly in FIGS. 5 and 9, the panel or wall 34 of the container 28 is formed at its lower portion with a laterally extending through slot 112. As formed, the slot constitutes a passage between the interior of the buffer containing chamber 24 and the interior of the gel-containing cavity 40. Overlying the slot and serving as, a seal thereagainst is a semi-permeable membrane or dialysis membrane 114 which is held in place by means of a strip, strips, or frame 120 mechanically secured to the wall or panel 34 by means of screws 124, as shown in FIG. 9. Suitable sealing beads 130 seated in cooperating grooves 134 abut the contacting face of the membrane 114 to ensure a fluid-tight seal. Thus, the semipermeable or dialysis-type membrane 114 provides a path of electrical conductivity between the interior of the buffer vessel 24 and the electrophoresis gel medium 44 contained in the cavity 40. At the same time, any passage of liquid or fluid between the container 24 and the cavity 40 is prevented. Although the fixed membrane structure is preferred, a removable mechanical barrier may be used instead.

As seen in FIGS. 1 and 3, the height of the wall 38 of the buffer container 28 is somewhat less than the height of the corresponding wall 34 of the buffer container 24. The reason for this structural arrangement is to permit the buffer solution which is introduced into the second container 28 to pour over the side wall 38 and to establish actual contact with the upper surface of the gel medium in the cavity 40. Recognizing that the gel medium constitutes a conductive path for the passage of charged particles, it will be appreciated that the membrane and fluid overflow system described above establishes electrical continuity between the buffer solution contained in each of the separate containers through the gel medium, and through the electrodes 92 and 102 and the associated lead lines 94 and 104 to the DC. power supply (not shown). For some techniques, it may be desired to separate the top of the gel from the liquid buffer solution in buffer container 28 by use of a semi-permeable membrane in a manner identical to that already described for the bottom of the gel.

As previously indicated, the apparatus of the invention is capable of being used to study and to analyze a plurality of samples simultaneously. In order to facilitate a reliable mechanical delineation from sample to sample, and as an aid to the positioning and location of the sample introduction along the gel medium 44, there is provided a comb-like pocket former which is illustrated in FIG. 10. In use, the pocket former occupies a position extending across the upper marginal portion of the gel-forming medium and projecting thereinto during the polymerizing or setting of that medium. The pocket former I40 is thereafter carefully removed to leave a lineal array of spaced sample receiving pockets, each readily accessible.

In the preferred embodiment of the apparatus.illustrated, the structural material used is plastic and preferably a transpatent plastic such as a polyacrylate (Lucite or Plexiglass). Any other plastic composition or other nonconductive structural material may be used. The preferred gel-forming medium has been described as an acrylamide. It will be appreciated, however, that any other material such as hydrolyzed starch may be used as a particular research program may warrant. Many of the other mechanical elements which have been described will appear clearly to be merely illustrative of the inventive concept disclosed and specific examples are not to be interpreted in any limiting sense. The apparatus itself is highly adaptable for both specialized and more general investigations and there is complete freedom of selection of not only the electrophoretic medium, but also of its shape and thickness, since a spacer of any required width may be used and since that spacer may be given any preferred contour. The preferred embodiment of the invention has been described as utilizing a permanently or semiperrnanently secured dialysis membrane. It will be appreciated that the type of membrane is readily selectable by the particular researcher for his particular purposes. Moreover, it is within the inventive concept of this development that a removable plug may be utilized in place of the semi-permeable membrane, although the membrane itself has many obvious and significant advantages over purely mechanical blocks or seals which must be removed prior to initiation of any electrophoretic procedure.

It is believed that the operation and use of the improved equipment of the invention will be apparent to those skilled in the art and, accordingly, only a brief general description is provided herebelow. After the containers or chambers 24 and 28 are clamped together with the spacer 30 in place, the gelling medium 44 is poured vertically, and the pocket former 140 is positioned in place at the top of the assembly. After polymerization or setting of the selected medium, buffer solution is poured into the cathodal chamber 28 until the solution covers the top of the gelled medium 44. The pocket former 140 is then removed directly without any necessity for excising. Buffer solution is then introduced into the anodal chamber or container 24 making electrical contact with the bottom region of the gelled medium through the dialysis membrane 114. Ions of the buffer solution now pass between the two containers 24 and 28, enabling current to be maintained, whileat the same time preventing the bulk passage of liquid. As an alternative technique, a sliding metal plate may be used as a temporary cover, gate, or closure to block the intercommunication between the anodal chamber and the base of the gel. The cover or gate actsto block objectionable liquid passage during the period the gel solution is fluid. After polymerization, the gate is raised to establish the necessary buffergel contact.

In filling all of the pockets formed in the gel, at the same time, it is convenient to use a multiple micro-syringe holder, this instrument consisting essentially of a plurality of ordinary syringe needles and actuators arranged as a linear array with spacing corresponding to the spacing established between gel pockets in the gel medium. A gauging bar extending under the plungers enables one to raise all plungers simultaneously to the desired selected volume which may be preset by thread controls on the guide rods. A second bar may be utilized to push the plungers down simultaneously. Alternatively, this operation may be done by hand. The microsyringe holder slips easily over guide rods on the gel box, ensuring that all needles will be correctly positioned over the gel pockets, every time.

An advantage achievable through the use of the apparatus of the present invention, as contrasted with prior art devices, is that the structure described and illustrated herein may be easily adapted for investigations in which a density gradient gel composition is employed. One such technique has been described by J. J. Pratt and W. G. Dangerfield in their article entitled Polyacrylamide Gels of Increasing Concentration Gradient for the Electrophoresis of Lipoproteins, Clin. Chim. Acta, 23 (1969) 189-201.

Many research programs directed to various facets of electrophoretic investigations which have been carried out are reported in the technical literature. A modified acrylamide electrophoresis method has been described by Robert F. Ritchie, John G. Harter and Theodore B. Bayles in J. Lab. & Clin. Med., Vol. 68, No. 5, November 1966, in article entitled Refinements of Acrylamide Electrophoresis. As reported, they used a chamber which was, in design and dimensions, similar to that described in R. C. Raymond et al. U. S. Pat. No. 3,208,929.

Thus, the present invention is directed to improved apparatus and methods for gel electrophoresis as that analytical tool was first used by Oliver Smithies as described in Biochemical Journal 61: 629 (1955), and in BiochemicalJoumal68: 636 (I958), 71: 585 (1959), and Adv. Protein Chem. 14: 65 (1959). The invention also sets forth new techniques for using acrylamide gel compositions, which materials were first used for electrophoresis by B. I. Davis as described in the article by Davis, Ornstein, Taleporos & Konlish, J. Histochem. Cytochem. 7: 291 (1959).

While disclosures of preferred embodiments of the apparatus of the invention and of preferred methods for assembly and installation of the apparatus have been provided, and while preferred structural materials and techniques have been indicated, it will be apparent that numerous modifications and variations thereof may be made without departing from the underlying principles of the invention. It is, therefore, desired by the following claims to include within the scope of the invention all such variations and modifications by which substantially the results of this invention may be obtained through the use of substantially the same or equivalent means.

What is claimed is:

' 1. A vertical gel electrophoretic apparatus in which the gel is set while disposed in a vertical attitude, said apparatus comprising:

first container means defining a first chamber for retaining buffer solution therewithin,

second container means defining a second chamber for retaining buffer solution therewithin,

panel means defining an elongate, vertically disposed cavity for retaining a gel-forming solution solidifiable in situ to form an electrophoresis gel medium, said panel means including wall means bounding said first and said second chambers,

said wall means bounding said first chamber having a through passage formed therein and communicating with said cavity,

fluid flow barrier means secured in said through passage between said first chamber and said cavity to preclude bulk passage of buffer solution between said first chamber and said cavity and to preclude passage of said gel-forming solution from said cavity to said first chamber prior to setting of said gel-forming solution,

said barrier means comprising an element of limited permeability and operable to permit passage of electrically charged particles therethrough but to preclude gross transfer of liquid between said first chamber and said cavity, and

means including electrode means within each of said first and said second chambers to facilitate transport of electrical current therebetween and through the gel medium contained in said cavity.

2. The structure as set forth in claim 1 wherein said barrier means comprises a sheet-like film having opposed principal surfaces presented respectively to said first chamber and to said cavity, whereby said film constitutes a separator therebetween.

3. An electrophoretic apparatus comprising a first chamber and a second chamber constituting a pair of buffer solution retaining vessels,

vertically extending wall means of corresponding said vessels being spaced laterally of and opposite one another to present said vertically extending wall means thereof in opposing and facing relation,

generally U-shaped upwardly opening spacer means interposed between said vessels and in liquid-sealing contact with the opposed facing wall means of said vessels to establish therebetween an upwardly opening cavity for receiving and retaining an initially fluid medium therewithin,

one of said vertically extending wall means having a through slot formed therein and extending thereacross,

barrier means secured in said slot,

said barrier means being of limited permeability to permit controlled passage of charged particles between said cavity and a chamber of one of said vessels through said slot, but to preclude mass passage of liquid therethrough, and

means including electrode means within said vessels to facilitate passage of current between said vessels and said cavity through media contained therein.

4. The structure as set forth in claim 3 wherein said vertically extending wall means have groove means formed therein at a lower portion and at opposed side marginal portions thereof,

and further comprising bead means seated in said groove means and extending outwardly therefrom along the length thereof to constitute gasket means for effecting a fluid-tight seal between said vertically extending wall means and said spacer means interposed therebetween.

5. A vertical gel electrophoretic apparatus in which the gel is set while disposed in a vertical attitude, said apparatus comprising:

first container means defining a first chamber for retaining buffer solution therewithin,

second container means defining a second chamber for retaining buffer solution therewithin, panel means defining an elongate, vertically disposed cavity for retaining a gel-forming solution solidifiable in situ to form an electrophoresis gel medium,

said panel means including wall means bounding said first and said second chambers,

slot means formed in and extending transversely through and across one of said wall means bounding one of said chambers,

fluid flow barrier means selectively positionable in said slot means to define a removable seal between said chamber and said cavity to preclude passage of said gel-forming solution from said cavity into said first chamber prior to setting of said gel-formin g solution in said cavity,

said barrier means being adapted for removal from said slot upon setting of said gel medium in said cavity thereby to permit entry of buffer solution into said slot to establish contact with said gel medium in said cavity and to provide an electrically conductive path between buffer solution in said first chamber and gel medium in said cavity, and

means including electrode means within each of said first and said second chambers to facilitate transport of electrical current therebetween and through the gel medium contained in said cavity.

6. The structure as set forth in claim 5 wherein respective said container means include vertical wall means adapted for disposition in a facing opposed parallel relationship,

and further comprising generally U-shaped upwardly opening spacer means positionable between said principal wall means to seal thereagainst at lower and side marginal portions thereof and to establish an upwardly opening cavity therebetween for receiving and retaining gel-forming solution therein.

7. The structure as set forth in claim 6 and further comprising cooperating clamping means carried by said container means to secure said first and said second container means in fixed cooperating relative positions and in stressing abutting engagement against said spacer means to ensure a positive fluid-tight seal between said spacer means and abutting said container means.

8. A vertical gel electrophoretic apparatus in which the gel is set while disposed in a vertical attitude, said apparatus comprising:

first container means defining a first chamber for retaining buffer solution therewithin,

second container means defining a second chamber for retaining buffer solution therewithin, panel means defining an elongate, vertically disposed cavity for retaining a gel-forming solution solidifiable in situ to form an electropheresis gel medium,

said panel means including wall means bounding said first and said second chambers,

slot means formed in and extending transversely through and across one of said wall means bounding one of said chambers, and

fluid flow barrier means extending across said slot to define a fluid-flow-restricting seal between said first chamber and said cavity to preclude passage of gel-forming solution from said cavity into said first chamber prior to setting of said gel-forming solution in said cavity.

9. An electrophoretic apparatus comprising a pair of upwardly opening, laterally spaced, facing and opposed vessels each adapted to retain buffer solution therewithin,

spacer means interposed between said vessels and in abutting, fluid-sealing engagement with facing walls thereof presented against said spacer means, thereby to establish an upwardly opening fluid retaining cavitybetween said vessels, said cavity being adapted to hold an in situ formed gel medium for electrophoresis studies,

fluid support means of limited permeability in one of said walls of said vessels to permit selective passage of particles therethrough between said one of said vessels and said cavity, and

means including electrode means in said vessels to permit current flow along a conductive path extending between said vessels and through said cavity interposed therebetween.

10. The structure as set forth in claim 1 and further comprising fluid passage means formed in said panel means for circulation of a heat exchange fluid therethrough to effectcooling of said panel means.

11. The structure as set forth in claim 3 and further comprising fluid passage means formed in said wall means for circulation of a heat exchange fluid therethrough.

12. The structure as set forth in claim 3 wherein said initially fluid medium within said cavity constitutes a gel-forming fluid which sets in situ within said cavity to provide a gel sheet bounded by said walls and whoe through thickness is dictated by the thickness of said spacer interposed between said opposed wall means of said vessels.

13. The structure as set forth in claim 3 and further comprising gasket means interposed between said wall means and said spacer means to ensure a fluid-tight seal therebetween.

14. The structure as set forth in claim 1 wherein said barrier means comprises a dialysis membrane.

15. The structure as set forth in claim 3 wherein said barrier means comprises a dialysis membrane.

16. The structure as set forth in claim 3 and further comprising clamping means for securing said spacer means against opposed said wall means in fluid-sealing, stressed relation therewith.

17. The structure as set forth in claim 1 wherein said element of limited permeability constitutes a removable mechanical barrier defining a separator between said first chamber and said cavity.

18. A vertical gel electrophoretic apparatus in which the gel is set while disposed in a vertical attitude, said apparatus comprising:

first container means defining a first chamber for retaining buffer solution therewithin,

second container means defining a second chamber for retaining buffer solution therewithin, panel means defining an elongate, vertically disposed cavity for retaining a gel-forming solution solidifiable in situ to form an electrophoresis gel medium,

said panel means including wall means bounding said first and said second chambers,

slot means formed in and extending transversely through and across one of said wall means bounding one of said chambers, and fluid flow barrier means extending across said slot to define a fluidflow-restricting seal between said chamber and said cavity to preclude passage of gel-forming solution from said cavity into said first chamber prior to setting of said gel-forming solution in said cavity.

Claims (17)

1. A vertical gel electrophoretic apparatus in which the gel is set while disposed in a vertical attitude, said apparatus comprising: first container means defining a first chamber for retaining buffer solution therewithin, second container means defining a second chamber for retaining buffer solution therewithin, panel means defining an elongate, vertically disposed cavity for retaining a gel-forming solution solidifiable in situ to form an electrophoresis gel medium, said panel means including wall means bounding said first and said second chambers, said wall means bounding said first chamber having a through passage formed therein and communicating with said cavity, fluid flow barrier means secured in said through passage between said first chamber and said cavity to preclude bulk passage of buffer solution between said first chamber and said cavity and to preclude passage of said gel-forming solution from said cavity to said first chamber prior to setting of said gel-forming solution, said barrier means comprising an element of limited permeability and opErable to permit passage of electrically charged particles therethrough but to preclude gross transfer of liquid between said first chamber and said cavity, and means including electrode means within each of said first and said second chambers to facilitate transport of electrical current therebetween and through the gel medium contained in said cavity.

2. The structure as set forth in claim 1 wherein said barrier means comprises a sheet-like film having opposed principal surfaces presented respectively to said first chamber and to said cavity, whereby said film constitutes a separator therebetween.

3. An electrophoretic apparatus comprising a first chamber and a second chamber constituting a pair of buffer solution retaining vessels, vertically extending wall means of corresponding said vessels being spaced laterally of and opposite one another to present said vertically extending wall means thereof in opposing and facing relation, generally U-shaped upwardly opening spacer means interposed between said vessels and in liquid-sealing contact with the opposed facing wall means of said vessels to establish therebetween an upwardly opening cavity for receiving and retaining an initially fluid medium therewithin, one of said vertically extending wall means having a through slot formed therein and extending thereacross, barrier means secured in said slot, said barrier means being of limited permeability to permit controlled passage of charged particles between said cavity and a chamber of one of said vessels through said slot, but to preclude mass passage of liquid therethrough, and means including electrode means within said vessels to facilitate passage of current between said vessels and said cavity through media contained therein.

4. The structure as set forth in claim 3 wherein said vertically extending wall means have groove means formed therein at a lower portion and at opposed side marginal portions thereof, and further comprising bead means seated in said groove means and extending outwardly therefrom along the length thereof to constitute gasket means for effecting a fluid-tight seal between said vertically extending wall means and said spacer means interposed therebetween.

5. A vertical gel electrophoretic apparatus in which the gel is set while disposed in a vertical attitude, said apparatus comprising: first container means defining a first chamber for retaining buffer solution therewithin, second container means defining a second chamber for retaining buffer solution therewithin, panel means defining an elongate, vertically disposed cavity for retaining a gel-forming solution solidifiable in situ to form an electrophoresis gel medium, said panel means including wall means bounding said first and said second chambers, slot means formed in and extending transversely through and across one of said wall means bounding one of said chambers, fluid flow barrier means selectively positionable in said slot means to define a removable seal between said chamber and said cavity to preclude passage of said gel-forming solution from said cavity into said first chamber prior to setting of said gel-forming solution in said cavity, said barrier means being adapted for removal from said slot upon setting of said gel medium in said cavity thereby to permit entry of buffer solution into said slot to establish contact with said gel medium in said cavity and to provide an electrically conductive path between buffer solution in said first chamber and gel medium in said cavity, and means including electrode means within each of said first and said second chambers to facilitate transport of electrical current therebetween and through the gel medium contained in said cavity.

6. The structure as set forth in claim 5 wherein respective said container means include vertical wall means adapted for disposition in a facing opposed parallel relationship, and further comprising generally U-shaped Upwardly opening spacer means positionable between said principal wall means to seal thereagainst at lower and side marginal portions thereof and to establish an upwardly opening cavity therebetween for receiving and retaining gel-forming solution therein.

7. The structure as set forth in claim 6 and further comprising cooperating clamping means carried by said container means to secure said first and said second container means in fixed cooperating relative positions and in stressing abutting engagement against said spacer means to ensure a positive fluid-tight seal between said spacer means and abutting said container means.

8. A vertical gel electrophoretic apparatus in which the gel is set while disposed in a vertical attitude, said apparatus comprising: first container means defining a first chamber for retaining buffer solution therewithin, second container means defining a second chamber for retaining buffer solution therewithin, panel means defining an elongate, vertically disposed cavity for retaining a gel-forming solution solidifiable in situ to form an electropheresis gel medium, said panel means including wall means bounding said first and said second chambers, slot means formed in and extending transversely through and across one of said wall means bounding one of said chambers, and fluid flow barrier means extending across said slot to define a fluid-flow-restricting seal between said first chamber and said cavity to preclude passage of gel-forming solution from said cavity into said first chamber prior to setting of said gel-forming solution in said cavity.

9. An electrophoretic apparatus comprising a pair of upwardly opening, laterally spaced, facing and opposed vessels each adapted to retain buffer solution therewithin, spacer means interposed between said vessels and in abutting, fluid-sealing engagement with facing walls thereof presented against said spacer means, thereby to establish an upwardly opening fluid retaining cavity between said vessels, said cavity being adapted to hold an in situ formed gel medium for electrophoresis studies, fluid support means of limited permeability in one of said walls of said vessels to permit selective passage of particles therethrough between said one of said vessels and said cavity, and means including electrode means in said vessels to permit current flow along a conductive path extending between said vessels and through said cavity interposed therebetween.

10. The structure as set forth in claim 1 and further comprising fluid passage means formed in said panel means for circulation of a heat exchange fluid therethrough to effect cooling of said panel means.

11. The structure as set forth in claim 3 and further comprising fluid passage means formed in said wall means for circulation of a heat exchange fluid therethrough.

12. The structure as set forth in claim 3 wherein said initially fluid medium within said cavity constitutes a gel-forming fluid which sets in situ within said cavity to provide a gel sheet bounded by said walls and whoe through thickness is dictated by the thickness of said spacer interposed between said opposed wall means of said vessels.

13. The structure as set forth in claim 3 and further comprising gasket means interposed between said wall means and said spacer means to ensure a fluid-tight seal therebetween.

14. The structure as set forth in claim 1 wherein said barrier means comprises a dialysis membrane.

15. The structure as set forth in claim 3 wherein said barrier means comprises a dialysis membrane.

16. The structure as set forth in claim 3 and further comprising clamping means for securing said spacer means against opposed said wall means in fluid-sealing, stressed relation therewith.

17. The structure as set forth in claim 1 wherein said element of limited permeability constitutes a removable mechanical barrier defining a separator between said first chamber and said cavity.

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