US 3290240 A
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Description (OCR text may contain errors)
E. J. NEREN Dec. 6, 1966 VERTICAL ELECTROPHORESIS APPARATUS WITH ADJUSTABLE MEDIA TUBE Filed March 14, 1962 2 Sheets-Sheet 1 INVENTOR fan .4 e0 1 N555 Dec. 6, 1966 E. J. NEREN 3,290,240
VERTICAL E LECTROPHORESIS APPARATUS WITH ADJUSTABLE MEDIA TUBE Filed March 14, 1962 2 Sheets-Sheet 2 unmui n IN VENTOR.
United States Patent ()fiice Patented Dec. 6, 1966 3,290,240 VERTICAL ELECTROPHORESIS APPARATUS WITH ADJUSTABLE MEDIA TUBE Edward J. Neren, 603 Wolf St., Philadelphia, Pa.
Filed lVIar. 14, 1962, Ser. No. 179,577
9 Claims. (Cl. 204-299) This invention relates to a vertical electrophoresis apparatus and has primary applicability to gel media, 21- though the apparatus may be advantageously employed with other packed supporting media such, for example, as glass, sols, sponge rubber, and cellulose, and has as its primary object the provision of a relatively compact apparatus of any suitable size in accordance with its particular use for the vertical electrophoresis of blood serum, spinal fluid, tissue proteins, plasma, enzymes, hemoglobins, nucleic acids, polysaccharides, amino acids, and other similar materials or samples for spectrograph-it: qualitative analysis.
A further object of the invention is the provision of an apparatus of this character which may be thermally controlled, preferably but not necessarily for continuous cooling by water or other suitable fluid with efficient removal of electrode induced heat, whereby higher voltages can be used and faster separations obtained.
An additional object of the invention is the provision of an apparatus of this character wherein gel containers in the form of circular tubes accommodating gel slugs or rectangular tubes accommodating gel slabs may be interchangeably employed.
It is to be understood that the term gel media, as employed herein, is to be interpreted as including other packed media for the support of samples to be subjected to electrophoresis.
A still further object of the invention is the provision of a device of this character which is readily adapted to accommodate differing lengths of .gel tubes, without modification to the apparatus.
Still another object of the invention is the provision of a device of this character wherein the molecular flow or migration in a vertical direction eliminates any possibility of electro-osmotic flow.
Still another object of the invention is the provision of a device of this character which may be used in conjunction with organic solvent systems, or organic buffer solutions, by the substitution of Teflon, for example, for plastic where such plastic is employed.
Still other objects reside in the combinations of elements, arrangements of parts, and features of construction, all as will be more fully pointed out herein-after. Still other objects will in part be obvious and in part be hereinafter described and shown in the accompanying drawing wherein:
FIGURE 1 is an elevational view of one form of apparatus constructed in accordance with the instant inventive concept, certain concealed parts thereof being indicated in dotted lines.
FIGURE 2 is an enlarged vertical cross-sectional view of a portion of the apparatus disclosed in FIG. 1.
FIGURE 3 is a sectional view taken substantially along the line 3-3 of FIG. 2 as viewed in the direction indicated by the arrows.
FIGURES 4, 5, 6, 7, and 8 are views showing in plan, various type-s of top and bottom plugs which may be employed in conjunction with the apparatus of FIG. 2.
FIGURE 9 is an enlarged sectional view taken substantially along the line 99 of FIG. 2.
FIGURE 10 is. a view similar to FIG. 9 but showing a modification thereof.
FIGURE 11 is a view similar to FIGS. 9 and 10 but showing a further modification.
FIGURE 12 is a similar sectional view of a. still further modified form of apparatus; and
FIGURE 13 is a sectional view taken substantially along the line 13-13 of FIG. 12 as viewed in the direction indicated by the arrows.
.Similar reference characters refer to similar parts throughout the several views of the drawing.
Having reference now to the drawings in detail, the apparatus of the instant invention is generally indicated at 20, and may comprise a vertical standard 21 mounted on a base 22, the standard carrying a supporting clamp or ring 23 which is attached to an apertu-red boss 24 for longitudinal movement on the upright 21, a set screw 25 being provided for securing the support 23- at the desired altitude. Supporting ring 23 surrounds and supports a temperature control tube 26 which may be formed of glass, Teflon, Plexiglas, Pyrex or the like, and which is provided with a fluid inlet 27 and outlet 28 for circulation of a medium such as water or other temperature controlled fluid, preferably, though not necessarily, for cooling purposes, through the container. The bottom of the container is closed by means of a stopper or plug 29, which in the illustrative embodiment shown in FIGURE 2, for example, is provided with a central opening 30 through which extends a glass or plastic connecting tube 31. The lower end of tube 31 extends into a receptacle 32 which comprises a lower electrode compartment containing electrolytic fluid 33 and provided with a negative electrode 34, from which a wire 35' extends for a purpose to be more fully described hereinafter.
The upper end 36 of connecting tube 31 is provided with a tubular joint or coupling sleeve 37 which is preferab-ly integral therewith and which is adapted to connect the tube 31 with a gel tube 38 which contains any suitable media such, for example, as starch gel, agar, sols, glass spheres, methylcellulose, sponge rubber or the like, as generally indicated at 39.
An upper joint or coupling sleeve 40' serves to connect the upper end of gel tube 38 with an upper connecting tube 41, which extends through a central aperture 42 in a relatively thick stopper or plug 43, which completely closes the top of coolant containing receptacle 26 and at the same time serves as a lower closure for an upper electrode compartment 44, containing an electrolytic solution 45, and having positioned therein an electrode 46 which is connected by means of a wire 47 to a common conductor 48 which contains also the wire 35 leading from the negative electrode 34. While electrode 46 as well as the electrode 34 are shown as comprising plate type electrodes, it will be understood that a ring electrode may be employed in both the upper and lower electrode cells, if desired.
As best shown in FIGURE 1, the common conduit 48 extends to a suitable control unit 49 which is provided with voltage, amperage, and wattage indicators 50, 5-1, and 52, respectively, as well as suitable control knobs 53, 54, and 55 for suitably regulating any of the electrical properties of the positive electrode 46 as well as the lower or negative electrode 34. The flow tubes 27 and 28 are connected to fluid pipe means 56 and 57 which pass through a suitable valve means interiorly or" the control unit (not shown), the temperature of the fluid being indicated by temperature indicator 58, and suitable valve means, of conventional design (not shown) being employed to regulate the circulation of the fluid into the container or receptacle 26.
In the use and operation of the device, the upper electrode cell 44, the stopper 43, and the connecting tube 41 are first assembled, together with the joint 40. The lower assembly comprised of connecting tube 31 and joint 37 is then assembled with plug or stopper 29.
The gel slug comprised of agar, starch, polyacrylamide,
or any other suitable conventional gel substance is then formed in a gel tube 38 which is then connected to either the upper joint 40 or the lower joint 37. In the event that it is connected, for example, to the upper joint 40, the entire upper assembly is fitted into the top opening of the coolant receptacle 26 in fluid tight relation. The lower assembly including the joint 37 is then connected to the lower end of the tube 38, and the plug 29 seated firmly in the lower end of the member 26. The assembly is then inverted, and the tube 31 filled with a suitable butler solution removing all air. When the assembly is reinverted and the end of tube 31 containing the butter solution is inserted into the electrolytic fluid in the lower electrode cell, the position of the assembly is set by means of the set screw 25 through the clamping ring 23. The upper assembly including the plug 43 is then positioned in the top of receptacle 26 and the tube 41 filled with buffer solution, again removing all air. The electrolytic solution 45 may then be positioned in the upper electrolyte cell 44.
A sample to be treated, such as hemoglobin, serum, protein, or other suitable material for analysis is then inserted by means of a pipette or fill tube 60 directly on top of the gel slug 61 in gel tube 38. It is to be noted that the sample to be tested must have a greater density than the butter solution, so that it will settle on the top surface of the gel slug. Electrodes 46 and 34 are then connected through a line 62 and control box or unit 49 to a suitable source of DC. current, noting the polarity of the electrodes. The receptacle 26 is then filled with cooling fluid, such as water or the like, and the current flow started.
This procedure causes molecular migration of the sample through the gel slug 61 in tube 38, and may be continued until complete stratification of the components is effected.
Normally, a plain open ended gel tube or tubes are employed, but in certain instances a sample having both positive and negative components may be required for testing, and for this purpose a central opening 63 may be provided, if desired, and which is normally closed by a plug 64. By this arrangement such a sample may be inserted in the gel slug at a point near the center, to permit molecular migration in both directions.
Upon the completion of the operation the gel slug 61 .may be readily removed from its tube 38 after the apparatus has been disconnected by removal of either the upper plug 43 or the lower plug 29 and its associated ele- 'ments, the tube being separated at either the joint 40 or the joint 37.
It will be noted that this arrangement permits the use of gel tubes of varying length, since the relative insertion of tube 41 through plug 43, or tube 31 through plug 29,
may be readily varied in accordance with the length of the gel tube 38.
In the foregoing the apparatus has been described in connection with a single centrally positioned cylindrical gel tube, and the plugs 43 and 29 have been designed to accommodate sucha single sample. It will, however, be
readily understood that many variations of this principle may be utilized. For example, in FIG. 4 there is disclosed a plug 70 which has an elongated opening 71 therein for the accommodation of a single rectangular gel tube to provide a rectangular slab of gel to facilitate analysis when taneously treated.
FIGURE 7 discloses another modified form of plug 79 having three peripherally disposed openings 80 therein, each of which contains a rectangular gel tube 81 disposed in perimetric relation.
The plug 82 of FIGURE 8 is also provided with radially disposed elongated slots or openings 83 for containing a plurality of gel tubes 84 of rectangular configuration.
It is to be noted that any of the plugs 70, 73, 76, 79 or 82 may be readily substituted for the plugs 43 and 29, the only requirement being that identical plugs be employed at both the upper and lower ends of the apparatus.
In those cases where a sample to be subjected to elec- I trophoresis contains both positive and negative components, a central boss 63 is provided on the gel tube 38, as previously described and as best shown in FIG. 9. In the event that a rectangular tube 81 is employed as shown in FIG. 10, a similar central outlet 81' provided with a plug 82 may be employed for the same purpose.
FIGURE 11 discloses a rectangular tube 85 provided with three aligned parallel central openings 86, 86', and 86" which are closed respectively by plugs 87, 88, and 89 to permit the introduction of a plurality of individual samples into the same gel slab.
FIGURES 12 and 13 disclose a still further modified form of construction wherein a tube 90 is provided with a transverse elongated slotted opening 91, which is adapted for the reception of filter paper 92 which is employed frequently in electrophoresis processes rather than the gel slugs or slabs. In the case of the structure of FIG'URES 12 and 13, the filter paper may be inserted in a razor cut in a gel fill of standard constituents.
While the receptacle 26 and its associated plugs 29 and 43 are described as, and preferably are, cylindrical, it will be understood that tubes and conforming plugs of square or polygonal cross-section may be employed, if desired. It will also be understood that the temperat ture regulating tube, the connecting tubes and sleeves and the supporting media may be comprised of any suitable material, such as glass, Pyrex, Teflon, Plexiglas, or the like.
From the foregoing it will now be seen that there is herein provided an improved apparatus for vertical electrophoresis which accomplishes all the objects of this in vention, and others, including many advantages of great practical utility.
As many embodiments may be made of this inventive concept, and as many modifications may be made in the embodiments hereinbefore shown and described, it is to be understood that all matter herein is to be interpreted merely as illustrative, and not in a limiting sense.
1. A vertical electrophoresis apparatus comprising a support, an open ended temperature regulating tube car ried by said support, a plug means having an aperture therein closing an upper 'end of said temperature regulating tube, a media tube means including media sample retaining material means therein, an electrolytic cell means connected to the outer end of said plug means, an adjustable connecting tube means extending through the aperture in said plug means and into said electrolytic cell means for pipette sample filling thereof, a connecting sleeve means extending on said connecting tube means to sealably engage said media tube means, a second apertured plug means closing the lower end of said temperature regulating tube, a second adjustable connecting tube means extending through the aperture in said second plug means to connect to the lower end of said media tube means and extend into an oppositely disposed second electrolytic cell means, a second sleeve means extending on the upper end of said second connecting tube means to sealably and adjustably engage the lower end of said media tube means, said connecting tube means with extending sleeve means being slidably adjustable to permit vertical adjustment of said media tube means within said temperature regulating tube maens and removal of said media tube means therefrom, upper and lower conduit means communicating with the upper and lower portions of said temperature regulating tube to permit circulating temperature regulating fluid through said temperature regulating tube, and electrode means for applying an electric potential to said electrolyte cells and through said supporting media tube.
2. The structure of claim 1 wherein a plurality of apertures are provided in each plug, each aperture containing a connecting tube with a connecting sleeve thereon, the connecting tubes extending through said plugs being aligned, and a supporting media tube extending between the connecting sleeves of aligned connecting tubes.
3. The structure of claim 2 wherein said apertures, connecting tubes, and supporting media tubes are circular to produce supporting media slugs.
4. The structure of claim 2 wherein said apertures, connecting tubes, and supporting media tubes are transversely elongated to produce supporting media slabs.
5. The structure of claim 1 wherein an intermediate opening is provided in said supporting media tube for the introduction of samples containing both positive and negative components, and a closure plug is provided for said intermediate opening.
6. The structure of claim 1 wherein said temperature regulating tube is a coolant tube and said coolant tube, connecting tubes, connecting sleeves, and supporting media tube are comprised of glass.
7. The structure of claim 1 wherein said temperature regulating tube is a coolant tube and said coolant tube,
References Cited by the Examiner UNITED STATES PATENTS 6/1961 Van Oss et al. 204-299 1/1963 Ladisch 204 OTHER REFERENCES Haglund et 31.: Acta. Chemica Scandinavica 1950, pages 957-962.
Heftmann: Chromatography Electrochromatography in Columns, pages 412-419, 1961.
Smithies: Biochemical Journal, volume 71, 1959, pages 585-587.
JOHN H. MACK, Primary Examiner.
MURRAY TILLMAN, JOHN R. SPECK, Examiners.
G. KAPLAN, J. BATIIST, E. ZAGARELLA,