US3764513A

US3764513A - Electrophoresis chamber

Info

Publication number: US3764513A
Application number: US00250424A
Authority: US
Inventors: C Saravis
Original assignee: Marine Colloids Inc
Current assignee: Marine Colloids Inc
Priority date: 1972-05-04
Filing date: 1972-05-04
Publication date: 1973-10-09
Anticipated expiration: 1990-10-09

Abstract

Electrophoresis apparatus with adjacent compartments arranged to hold buffer solution, means for supporting in position an impervious backing member having a gel layer on its upper surface, a cover with an aperture for applying samples and a layer of bibulous material fixed to the cover so as to be in contact with gel and the buffer solution when the cover is in position.

Description

O i 1 United States Patent 1 [111 3,764,513 Saravis Oct. 9, 1973 [5 ELECTROPHORESIS CHAMBER 3,407,133 10/1968 Oliva et a1. 204/299 3,494,846 2/1970 Arquembourg..... [751 Invent sm'avlsi Mass' 3,674,678 7/1972 Post, Jr. et al 204/299 [73} Assignee: Marine Colloids, lnc., Rockland,

M i Primary Examiner-John H. Mack Assistant ExaminerA. C. Prescott [22] Flled: May 1972 Attorney-R. W. Furlong [21] Appl. No.: 250,424

[57] ABSTRACT 52 us. Cl. 204/299, 204/180 G Electrophoresis apparatus with adjacent p 51 int. Cl B01k 5/00 merits arranged to hold buffer solution, means for p- [58] Field of Search 204/180 s, 180 G, porting in position on impervious backing member 204/299 300 having a gel layer on its upper surface, a cover with an aperture for applying samples and a layer of bibulous 5 Refe Ci material fixed to the cover so as to be in contact with UNITED STATES PATENTS gel and the buffer solution when the cover is in posit R24,752 l2/l959 Ressler 204/180 G Ion 3,371,027 2/1968 LaPaglia et al. 204/299 6 Claims, 4 Drawing Figures PAIENIEU mm 9197;

SHEET 10F 2 FIG I ELECTROPHORESIS CHAMBER This invention relates to electrophoresis, including immunoelectrophoresis, and pertains more specifically to improved apparatus for conducting a variety of electrophoretic reactions or separations.

Electrophoretic reactions or separations are conducted in thin layers of gel arranged to be in contact with electrically conductive liquids such as buffer solutions in which are immersed electrodes. The material to be reacted or separated is normally applied to the gel layer while an electric potential is applied to the electrodes. Because of the varying mobility of the molecules of the test material, certain of them are separated from others and/or are caused to enter into biophysical reactions in the gel layer. Because of the toxic or infectious nature of many of the materials employed, it is important that the operator or individual carrying out the procedure avoid contact with the materials as well as with the buffer solution itself.

The present invention provides apparatus for carrying out electrophoretic reactions and separations constructed and arranged so that the apparatus can be used repeatedly with minimum risk of the operator coming into contact with the test specimens or with any portion of the apparatus or of the electrical circuit with which the specimens or the buffer solution comes into contact. The procedures for using the apparatus and of replacing the gel layer after use are simple and efficient, facilitating use of the apparatus by operators with a minimum of training. The device is useful for all kinds of electrophoresis including, among others, counterelectrophoresis and immunoelectrophoresis.

In the drawings,

FIG. 1 is a view in cross-section showing one embodiment of the present invention;

FIG. 2 is a view in longitudinal section taken along line 2--2 of FIG. 1;

FIG. 3 is an exploded view in longitudinal elevation, partly broken away and in section, showing another embodiment in the process of assembly; and

FIG. 4 is a view in cross-section showing the embodiment of FIG. 3 after assembly.

As shown in the drawing, the apparatus of FIGS. 1 and 2 comprises a container 10 separated into two compartments by an imperforate median baffle 12. The container and baffle are preferably constructed of a synthetic plastic material which is both electrically insulating and resistant to attack by the buffer solutions it is intended to contain. Extending across each compartment adjacent baffle 12 is a cylindrical mandrel l4, 16 also of electrically insulating plastic material on which is wound a spiral

platinum wire electrode

18, 20 extending to

terminals

22, 24 arranged to be connected to a suitable source of direct current (not shown). The spiral configuration of the wire facilitates elimination of bubbles which might otherwise interfere with conductivity. The electrodes are arranged to provide a short electrical path from one to the other during use.

Also extending across each compartment are

additional baffles

26, 28, 30, 32, each provided with

slots

34, 34 adjacent top and bottom margins to provide for element 36, being supported by

baffles

26, 28, 30, and 32 is always maintained at the desired level in the container and serves as a firm base for maintaining the gel layer and its carrier in fixed position during application of the test material and operation of the device, ensuring uniform test conditions. The plastic backing member can be suitable water insoluble material such as a vinyl or acrylic polymer but is preferably transparent and preferably a polyester such as polyethylene terephthalate sold under the trade name Mylar or Cronar. The gel layer can be cross-linked dextran, polyacrylamide, or any other gel commonly used for this purpose, but is preferably agar or agarose. It may be a freshly prepared aqueous gel layer or one which has been previously dried and which is rehydrated either before or after being placed in the apparatus. When the backing member is transparent, the supporting element 36 is preferably opaque and black or other dark color to facilitate visual inspection of the gel layer during operation of the apparatus. Cover 42 which seats upon the walls of container 10 is provided with an aperture 44 positioned above gel layer 40 and has secured to its bottom adjacent opposite sides of aperture 44 a layer of bibulous material 46 from about 56 to V4 inch thick in position to bear upon each opposing margin of gel layer 40 and extending laterally away from aperture 44 to a position beyond the margin of the gel layer. The layer of bibulous material may be any hydrophilic material such as cotton fabric, filter paper, regenerated cellulose open-celled sponge, or the like, capable of acting as a wick for the electroconductive liquid. Each layer of bibulous material thence extends downwardly supported by a rigid reinforcing plastic member 48 through appropriate slots 50 in support element 36. In the embodiment shown, the apparatus is designed to accept backing member 38 in the form of a narrow elongated strip; additional slots 52 may be provided in support element 36 to accomodate other covers having layers of bibulous material more widely spaced so as to accomodate backing members and gel layers of different sizes and shapes. Sprocket 54 is mounted on an external wall of container 10 and take-up sprocket 56 is mounted at the opposite end of container 10, being provided with crank handle 58, and arranged to engage with appropriate sprocket holes in the margin of backing member 38.

In operation of the device, the compartments are filled with suitable electroconductive liquids, e.g., buffer solutions such as an aqueous solution of sodium barbital to a level not exceeding that of support member 36. The support element 36 is then put in place on top of the baffles and a strip of backing member carrying on its upper face a suitable layer of gel in a supplying reel (not shown) is led around sprocket 54 across the upper surface of support element 36 and over sprocket 56. Cover 42 is then put in place with the layers of bibulous material 46 extending into the buffer solutions on opposite sides of baffle l2 and serving as a wick to transport the buffer solution into contact with the opposing margins of gel layer 40, whence it permeates completely across the gel layer to provide an electroconductive path from one electrode to the other. The construction of the apparatus allows the wicks 46 to be applied simultaneously and uniformly to the margins of the gel layer 40 and avoids the necessity for the hands or fingers of the operator to come into contact with wicks 46 at any time. By employing a suitable adhesive for securing the wicks to the bottom of cover 42 it is possible to remove and replace the wicks when and if desired. Moreover, because the level of the buffer solution in the compartments is below the level of gel film 40 on backing member 38, and because slots 50 are wider than the combined thickness of bibulous material 46 and reinforcing member 48, there is no possibility of a short circuit developing beneath the gel film. Measured quantities of the material to be tested are placed in spaced locations on gel layer 40 through aperture 44, and the electrodes are connected to a suitable source of low voltage direct current (about 25 volts). After the desired reaction or separation has occurred, as may be determined for example by visual inspection through aperture 44 against the black background of supporting element 36, crank handle 58 is used to advance the strip of backing material with the attached gel layer, the used gel layer stored if desired while the fresh layer is advanced beneath aperture 44 on top of support member 36. Additional test materials are then introduced in the same manner.

In the embodiment shown in FIGS. 3 and 4, open topped container 100 of synthetic plastic material is provided with a centrally-disposed imperforate baffle 102 dividing the container into two liquid-tight compartments. Each end wall 104, 105 of the container is provided with a recessed shoulder 106 adapted to support removably an end of supporting element or bridge member 108 which serves to support on its upper face,

backing member 38 with its gel film 40. Bridge member 108 also supports two spaced apart

baffies

110, 112 arranged to be on opposite sides of baffle 102 when the ends of the bridge member are supported on

shoulders

106, 106. Each baffle carries on its electrically nonconductive face nearest baffle 102 an

electrode

114, 116 of a metal such as silver or platinum in the form of a thin narrow film or ribbon deposited for example by electroplating, sputtering or similar technique and bonded to the face of the baffle. In one end of supporting element 108 are mounted male bayonet

electrical connectors

118, 118 each connected to an end of one

electrode

114, 116 by a

suitable wire conductor

120, 120. Mounted in end wall 105 above shoulder 106 is a pair of

female connectors

122, 122 in position to receive detachably the

connectors

118, 118 when the bridge member is in position supported by

shoulders

106, 106.

Connectors

122, 122 are connected by ap propriate wires to a suitable source of direct electrical current (not shown). A slot 124 is provided near the upper edge of each

baffle

110, 112 above

electrodes

114, 116 to permit adequate circulation of buffer or electrolyte solution and provide a short electroconductive path from each electrode, and the lower end portions of

baffles

110, 112 are cut away to permit insertion and removal of the bridge member as shown in FIG. 3. A pair of

slots

126, 126 is provided in bridge member 108 each disposed laterally outwardly of baffles I10, 112 respectively to permit a wick to pass through it into contact with the liquid.

Cover 130 the edges of which are received and supported by shoulders 132 inset in the walls of container 100 is provided with a central aperture 134 in position to overlie gel film 40 on supporting element 108 when the device is assembled and is otherwise similar to the cover 42 of the first embodiment, being provided with two wicks of

bibulous material

136, 136 supported on reinforcing

members

138, 138 secured to the bottom of cover in position to extend through

slots

126, 126 of supporting element 108, and having their adjacent upper edges spaced from each other in position to lie in contact with opposing marginal portions of gel film 40.

A closure 140 having upstanding handles or

finger grips

142, 142 is arranged to fit into aperture 134 of cover 130 to seal it against dust and to reduce evaporation of electrolyte during use.

In use of the device the two compartments are filled to the desired level with suitable buffer solution and supporting element 108 is placed in position while joining

connectors

118, 122 as shown in FIG. 3. Gel film 40 with its backing 38 may be mounted in position along the center of the upper surface of supporting element 108 either before or after the supporting element is put in place, whereupon cover 130 is positioned with its edges supported by shoulders 132 and with the margins of

wicks

136, 136 in overlying contact with opposing marginal portions of gel film 40. The dimensions and clearances of the various components of the device are such that when supporting element 108 is in place with its ends resting on shoulders 106 and with

connectors

118 and 122 engaged, the lower surfaces of wicks 136, which are resilient after swelling with liquid buffer solution, are in gentle but firm contact with the upper marginal surfaces of gel film 40, also swollen with the liquid, thus providing a continuous electrical path from one electrode to the other by way of the buffer solution, the wicks and the gel film.

Samples to be tested are placed on the surface of gel film 40 in the desired locations and are subjected to analysis by application of an appropriate electrical current to the electrodes, as in the embodiment of FIGS. 1 and 2. Some or all of the container, bridge member, cover, and closure can be made of transparent plastic material to facilitate observation of the sample and of the interior of the container. Desirably the surface of supporting element 108 directly beneath gel film 40 on its backing member 38 can be opaque and of some color such as black to provide a contrast of color of the sample and to facilitate observation of the latter.

It will be appreciated that the construction of the present invention facilitates changing the buffer solution and the gel layer with a minimum risk of contamination to the operator because there is no necessity of touching or handling the bibulous materials or the buffer liquid.

I claim: 1. In electrophoresis apparatus comprising a pair of adjacent compartments each constructed and arranged to hold a supply of buffer solution,

an electrode in each said compartment in position to be submerged in the respective supply of buffer solution and means for connecting said electrodes to a source of electrical potential, and

means for supporting in position above the surface of said supplies of buffer solution an impervious backing member having an electrophoresis gel layer on its upper surface,

the improvement comprising a cover for said compartments having an aperture positioned over the location of said gel layer arranged to permit introduction therethrough of material to be subjected to electrophoresis, said cover having secured to its bottom adjacent each opposite side of said aperture a layer of bibulous material in position to bear upon each opposing margin of said gel layer, each said layer of bibulous material extending laterally away from said aperture to a position beyond the margin of said underlying gel layer, thence downwardly into one of said compartments, each said layer of bibulous material being constructed and arranged to come into contact with its respective supply of buffer solution.

2. Apparatus as claimed in claim 1 including means for advancing said backing member with its gel layer along said supporting means.

3. Apparatus as claimed in claim 1 in which said supporting means for said backing member extends over said compartments and is provided with slots through which said layer of bibulous material extends downwardly.

4. In electrophoresis apparatus comprising a pair of adjacent compartments each constructed and arranged to hold a supply of buffer solution,

the improvement comprising removable means for supporting in position above the surface of said supplies of buffer solution an impervious backing member having an electrophoresis gel layer on its upper surface, said supporting means including a pair of electrodes and means for supporting each said electrode in each of said compartments in position to contact the respective supply of buffer solution and means for detachably connecting said electrodes to a source of electrical potential, and a cover for said compartments having an aperture positioned over the location of said gel layer arranged to permit introduction therethrough of material to be subjected to electrophoresis said cover having secured to its bottom adjacent each opposite side of said aperture a layer of bibulous material in position to bear upon each opposing margin of said gel layer, each said layer of bibulous material extending laterally away from said aperture to a position beyond the margin of said underlying gel layer, thence downwardly into one of said compartments, each said layer of bibulous material being constructed and arranged to come into contact with its respective supply of buffer solution.

5. Apparatus as claimed in claim 4 in which the means for supporting each said electrode comprises a baffle extending downwardly into each compartment.

6. Apparatus as claimed in claim 5 in which the detachable electrical connection means comprises a connection between said support means and the wall of the compartment.

Claims

1. In electrophoresis apparatus comprising a pair of adjacent compartments each constructed and arranged to hold a supply of buffer solution, an electrode in each said compartment in position to be submerged in the respective supply of buffer solution and means for connecting said electrodes to a source of electrical potential, and means for supporting in position above the surface of said supplies of buffer solution an impervious backing member having an electrophoresis gel layer on its upper surface, the improvement comprising a cover for said compartments having an aperture positioned over the location of said gel layer arranged to permit introduction therethrough of material to be subjected to electrophoresis, said cover having secured to its bottom adjacent each opposite side of said aperture a layer of bibulous material in position to bear upon each opposing margin of said gel layer, each said layer of bibulous material extending laterally away from said aperture to a position beyond the margin of said underlying gel layer, thence downwardly into one of said compartments, each said layer of bibulous material being constructed and arranged to come into contact with its respective supply of buffer solution.

4. In electrophoresis apparatus comprising a pair of adjacent compartments each constructed and arranged to hold a supply of buffer solution, the improvement comprising removable means for supporting in position above the surface of said supplies of buffer solution an impervious backing member having an electrophoresis gel layer on its upper surface, said supporting means including a pair of electrodes and means for supporting each said electrode in each of said compartments in posiTion to contact the respective supply of buffer solution and means for detachably connecting said electrodes to a source of electrical potential, and a cover for said compartments having an aperture positioned over the location of said gel layer arranged to permit introduction therethrough of material to be subjected to electrophoresis said cover having secured to its bottom adjacent each opposite side of said aperture a layer of bibulous material in position to bear upon each opposing margin of said gel layer, each said layer of bibulous material extending laterally away from said aperture to a position beyond the margin of said underlying gel layer, thence downwardly into one of said compartments, each said layer of bibulous material being constructed and arranged to come into contact with its respective supply of buffer solution.