US3375187A

US3375187A - Apparatus for temperature controlled preparative and analytical electrophoresis

Info

Publication number: US3375187A
Application number: US455401A
Authority: US
Inventors: Buchler Joseph
Original assignee: Buchler Instruments Inc
Current assignee: Buchler Instruments Inc
Priority date: 1965-05-13
Filing date: 1965-05-13
Publication date: 1968-03-26
Anticipated expiration: 1985-03-26
Also published as: FR1499413A

Description

March 26, 1968 .1. BUCHLER 3,375,187

APPARATUS FOR TEMPERATURE CONTROLLED PREPARATIVE AND ANALYTICAL ELECTROPHORESIS Filed May 13, 1965 4 Sheets-Sheet l Specfro- Phofomefer F Joseph Buchler JNVENTOR.

March 26, 1968 J. BUCHLER 3,375.18?

APPARATUS FOR TEMPERATURE CONTROLLED PREPARATIVE AND ANALYTICAL ELECTROPHORESIS Filed May 13, 1965 4 SheetsSheex I N VEN TOR.

Attorney Joseph Buchler' March 26, 1968 J.- BUCHLER 3,375.18

APPARATUS FOR TEMPERATURE CONTROLLED PREPARATIVE AND ANALYTICAL ELECTROPHORESIS Filed May 13, 1965 4 Sheets-Sheet 5 Joseph Buchler INVENTOR.

BY A

Attorney 3,375,187 D PREPARATIVE 13515 March 26, 1968 J Uc APPARATUS FOR TEMPERATURE CONTROLLE A'ND ANALYTICAL ELECTROPHOR Filed May 13, 1965 4 Sheets-Sheet 4 Fig.4

' Y Josph Buchler INVENTOR. 7

BY 5S Attomy United States Patent 3,375,187 APPARATUS FOR TEMPERATURE CON- TROLLED PREPARATIVE AND ANA- LYTICAL ELECTROPHORESIS Joseph Buchler, Fort Lee, N.J., assignor to Buchler Instruments, Inc., Fort Lee, N.J., a corporation of the United States Filed May 13, 1965, Ser. No. 455,401 16 Claims. (Cl. 204-301) My present invention relates to an improved apparatus for temperature-controlled preparative and analytical electrophoresis and, more particularly, to gel-electrophoretic systems.

It has recently been suggested that considerable improvement in the resolving power of electrophoresis processes can be accomplished in a gel-column system, advantageously with the use of a polyacrylamide medium in a temperature-controlled electrophoresis apparatus. In fact, T. Jovin, A. Chrambach and M. A. Naughton have proposed a preparative-electrophoresis column in which effective temperature control can be maintained (Analytical Biochemistry, vol. 9, No. 3, November 1964, page 351). The basic elements of this system include a buffer reservoir terminating at an electrophoresis column which is surrounded by a temperature-control jacket through which a cooling liquid is circulated, an elution tube passing through this column and terminating at an elution chamber between a membrane and the gel column.

My present invention relates to such systems and particularly to improvements therein whereby the apparatus can be assembled and disassembled without the need for personnel skilled in glass-blowing procedures, which renders the apparatus substantially more comp-act than possible heretofore, and which dispenses to a large measure wtih problems resulting from air inclusion, etc., in earlier apparatus setups.

It is thus the principal object of the present invention to provide an improved apparatus for preparative gel electrophoresis which is simpler to operate and assemble, is more effective and is less complicated than has been possible heretofore.

A further object of this invention is to provide improved means, in an apparatus of the character described, for supporting the electrophoresis column and adjusting same.

These objects and others, which will become apparent hereinafter, are attained, in acordance with the present invention, by providing, in an apparatus having an electrophoresis column containing the gels and surmounted by a reservoir for the buffer solution, a tubular membrane holder designed to extend into this column and removably received within a column-support collar by means of which the column can be mounted upon a base. Advantageously, the cylindrical membrane-support for the continuous-flow electrophoresis apparatus is provided with annular sealing means .co-operating with the collar to provide a liquid-tight assembly therewith and has the membrane removably .mounted therein by an annular locking means. The membrane support is, according to this invention, a substantially self-contained unit adapted to be filled with a conductive liquid and closed on one end by the membrane and on the other end by a further membrane or screen. The use of flexible membranes on both ends of the tubular diaphragm holder permits hydrostatic equilibration by controlling the liquid head of of the buffer solution surrounding the lower membrane. Since, however, the elution buffer is of fluctuating pressure when displaced by a pulsating or peristaltic pump, I prefer to employ a rigid diaphragm in juxtaposition with the gels and to define the gap from which the elution buffer is withdrawn.

According to a more specific feature of the present invention, the gel column is composed of glass and is provided with a cooling jacket which extends beyond the gel-containing portions of the column so as to surround the membrane and at least an upper portion of the tubular support therefor, the collar comprising a pair of threadedly interconnected annular members of which one is affixed to the glass column while the other receives the membrane support. This arrangement permits the membrane support to be removed from the column and the column to be detached from its base without the need for complete disassembly procedures and permits the parts to be readily assembled. The collar consists of these annular members and the membrane-support tubes preferably are composed of a transparent synthetic resin (e.g., an acrylic polymer) while the seals are formed by O-rings received in annular grooves in the mating parts. The collar itself can be seated in an annular base forming the reservoir for the lower buffer and one of the electrophoresis electrodes. The jacket, moreover, includes improved heat-exchanger means for the elution buffer to ensure temperature equilibrium within the system.

According to another feature of the present invention, the buffer reservoir is provided with improved means for adjustably supporting the elution capillary and the cooling sleeve surrounding same.

In prior apparatus for gel electrophoresis, adjustment of the distance between the lower end of the cooling finger by means of which the buffer is eluted was diflicult, if not impossible, and the dimensions of the cooling finger were critical. I have found, however, that the cooling finger, which is generally provided with a standard tapered joint, can be adjustably accommodated in the open end of the upper buffer reservoir by an assembly which basically comprises an externally threaded sleeve having a corresponding female tapered joint; an internally threaded adapter mounted upon the buffer reservoir and adjustably receiving this sleeve; and a locking ring threaded onto the sleeve and securing it in place in various positions of adjustment. When the buffer reservoir is provided with a standard tapered joint, the internally threaded adapter advantageously has a male taper corresponding thereto. It is frequently desired, however, to provide the buffer reservoir with a relatively wide-mounted cylindrical boss. In this case, the adapter is advantageuosly a cap formed with an annular recess designed to receive the lip of the boss and positively position the cap upon the boss. When it is desirable to mount an appliance (such as a thermometer, an electrode, etc.) in the buffer reservoir, the adapter can be provided with a suitable passage or bore for this purpose. Since ease of manipulation requires that the locking ring be of a relatively large diameter, it may be provided with an opening, according to this invention, aligned with the passage in the locked position of the ring so as not to obstruct this passage.

As previously noted, it is an important feature of this invention that the membrane proximal to the gel and defining therewith the elution chamber be rigid so as to eliminate the need for hydrostatic equilibration between the upper and lower buffers or at least to reduce the sensitivity of the system to hydrostatic fluctuation. This is accomplished by using, as the membrane, a porous glass plate which can be held in place in a suitable recess by a ring or the like. Advantageously, this membrane and the membrane on the opposite extremity of the cylindrical support are held flush with the latter by suitable locking rings.

The above and other objects, features and advantages of the present invention will become more readily apparent from the following description, reference being made to the accompanying drawing in which:

FIG. 1 is a vertical cross-sectional view through a continuous-flow electrophoresis apparatus, diagrammatically illustrating the auxiliary equipment for carrying out electrophoresis;

FIG. 2 is a perspective exploded view of the bottom of the column and its support; 1

FIG. 3 is a perspective exploded view of the top of the column showing the adjusting means for the elution tube;

FIG. 4 is a vertical cross-sectional view through a membrane holder according to another embodiment of the present invention;

FIG. 5 is a vertical cross-sectional view through another adjustment means according to this invention; and

FIG. 6 is a plan view of this latter structure.

In the drawing I show an electrophoresis assembly which comprises an electrophoresis column 10 with a precision internal bore 11 in which upper and

lower gels

12, 13 are provided as described by T. Jovin et al. (supra). The electrophoresis column 10 is surmounted by a buffer reservoir 14 having a ground-glass tapered joint 15 into which the material to be subjected to electrophoretic resolution can be introduced and receiving a platinumwire electrode 16 sealed in a glas electrode holder 17 with a complementary tapered joint. The terminal 18 of the electrode holder can be connected to the output terminal of a conventional high-voltage electrophoresis power supply in the usual manner.

The buffer reservoir 14 is provided with a wide-mouth ground-glass tapered joint 19 through which the upper buffer can be introduced and in which a cold finger 26 is adjustably mounted as will become apparent hereinafter. The cold finger contains a central elution capillary 21 surrounded by a cooling jacket 22 whose inlet 23 and out let 24 are connected in a coolant-circulating path in the usual manner. The elution capillary 21 has an outlet 25 through which the elution buffer can be withdrawn. As can be seen from FIG. 1, the elution capillary 21 has a mouth 26 terminating at the lower surface of the

gel layers

12, 13 above a porous glass membrane 27 which is slightly spaced from the gels to form an elution compartmcnt 28. The electrophoresis column 10, which is advantageously composed of glass, is integral with a cooling jacket 29 which extends beyond the

layers

12, 13 of the gel to define a compartment 30 into which the membrane holder 31 extends. The jacket 29 has an inlet 32 for the coolant, a downwardly turned finger 33 insuring that the coolant flows into the regions of the jacket surrounding the chamber 30 and within a threaded ring 34 forming part of the collar of the present invention. The jacket 29 is further provided with an outlet 35 for the coolant and receives a heat-exchange coil 36 through which the elution buffer is fed from a nipple 37 extending through a wall of the jacket 29 and sealed thereto. The heat-exchanger coil 36 communicates with the clearance 30, surrounding the membrane holder 31, whereby the elution buffer is fed to the chamber 28 from all sides around the

gels

12, 13 and can be withdrawn centrally from the chamber 28 by capillary 21. A further tube 38 communicates with chamber 28 at the face of the gel layer 13 to enable entrapped air to be withdrawn upon assembly of the apparatus.

According to a particular feature of the present invention, the lower end 39 of the jacket 29 is received within the collar member 34 which is roughened along internal surface 40 to insure firm bonding with the glass jacket 29. An adhesive 41 secures the ring 34 to this jacket. Advantageously, an upstanding boss 42 is provided centrally in the lower collar member 43 to project limitedly into the clearance 30 when the collar member 34 is threaded into member 43. Collar member 43 is internally threaded at 44 to receive a complementarily threaded portion 60 of membrane holder 31 and is formed with a ledge or shoulder 45 adapted to rest upon a base 46 forming a reservoir for the lower buffer. Shoulder 45 is provided with an annular recess for an O-ring 47.

Clamping means in the form of screw-type clamps 48 hold the

collar assembly

34, 43 onto the base 46. The latter communicates with vertically extending tube or standpipe 49 constituting a liquid column when filled with the buffer solution to provide hydrostatic equilibrium across the diaphragm 27. Another platinum electrode 50 is disposed in the interior of the base 46 below the membrane in holding sleeve 31 to provide another terminal to which the electrophoresis power supply can be connected.

From FIG. 1 it may be seen that the sleeve 31 is provided with the porous glass membrane 27 and a liquid permeable membrane or screen 51, the later being held in place by a locking ring 52 received within an annular recess 53 at the enlarged bottom end 54 of the membrane holder 31. This enlarged bottom end forms a manually engageable boss by means of which the membrane holder can be rotated to thread it into the column member 43. For this purpose, the boss 54 can be fluted, knurled or otherwise roughened to prevent slippage, Holder 31 is externally provided with

annular grooves

56, 59 to receive O-

rings

57, 58.

The membrane holder 31 is provided with a generally cylindrical upper portion while the membrane or screen 51 is stretched tightly by the locking ring 52 flush with the lower end of the holder. A rubber O-ring 62 locks the glass membrane is place. It should also be understood, however, that a shaped or profiled synthetic-resin ring can be substituted for the O-ring 62 to hold the glass membrane 27 in place.

The means for adjusting the location of the inlet end of the elution capillary 21, i.e. its spacing above the membrane 27, includes, according to my invention, an adapter 63 mounted on the mouth 19 of the upper bulfer reservoir 14 and provided with internal threads 64. An externally threaded sleeve 65 can be screwed into the adapter 63 to permit adjustment of the height of the cold finger 20 which is received in the sleeve 65. For this purpose, the sleeve 65 is formed with a female taper 66, complementary to the male taper 67 of the cold finger 20. A locking ring 67 is threaded onto the sleeve 65 and, upon rotation of the sleeve 65 to adjust the height of the cold finger, this locking ring 67 is tightened against the head of the adapter 63. The head or flange of the sleeve 65 can be fluted, milled or otherwise roughened to permit a secure grip during adjustment, while the nut 67 can be similarly roughened to afford ease of locking of this assembly. In general, the nut 67 should have a diameter exceeding that of the sleeve 65. In the structure of FIGS. 1 and 2, the open mouth of the buffer reservoir 14 is a tapered female joint 19 and, accordingly, the adapter 63 is provided with a complementary male taper.

The apparatus described above is operated substantially as discussed by T. Iovin et al. (supra) and the operation will be only briefly summarized here. For perparative polyacrylamide gel electrophoresis, a pair of gel layers 12, 13 are formed in the precision bore 11 of the electrophoresis column and the latter with its upper collar member 34 is threaded into the lower collar member 43 carrying the membrane holder 31. The membrane holder is adjusted to provide approximately 1 mm. of clearance between the lower gel surface and the membrane 27 by rotation of the membrane holder in the lower column member 43 to which it is threadedly secured. The assembly is then mounted upon the base 46 which is filled with buffer, additional bulfer being placed in the upper butler reservoir 14; the level of the buffer in this reservoir and that of the standpipe 49 are maintained identical to insure hydrostatic equilibrium. A coolant is circulated through the jacket 29 and the cooling finger 22 surrounding the capillary 21, the latter having been adjusted via the sleeve 65 so that its lower extremity is flush with the bottom surface of the

gels

12 and 13. The sample to be subjected to electrophoresis resolution and separation is placed at the upper surface of the gel 12 as described by T. Jovin-et al. and the electrophoresis voltage is applied across the electrodes 16 and 50. The elution butler enters through inlet 37 and is brought to temperature equilibrium in the heat exchanger 36 before it enters the clearance 30 and flows annularly into the clearance 28. Outlet tube 38 serves to remove any entrapped air at the lower surface of the gel column and can be closed via a stopcock or clamps after all gaseous inclusion have been sweep from the compartment 28 by the liquid stream. As the rings of the substance subjected to electrophoresis pass along the gel column downwardly, they are resolved (i.e. separate axially) and eventually are carried by the elution butter into the elution capillary 21 from which they are drawn by a peristaltic (finger) pump or the like into a detecting instrument such as a recording spectrophotometer.

In FIG. 4, I show a modified membrane holder in which the membrane 70 is flexible rather than rigid as is the case with membrane 27. The membrane 70 is held by a locking ring 71 taut across the opening of the membrane holder 72. The latter is otherwise identical with the membrane holder 31. Whereas the use of a rigid membarne 27 eliminates problems arising from pulsating flow of the elution buffer and reduces the sensitivity of the system to hydrostatic disequilibration, a flexible membrane 70 requires that care be taken in insuring that the upper and lower buffers are at hydrostatic equilibrium.

In FIGS. 5 and 6, I show a modified means for adjusting the cold finger and its capiliary 21. In this arrangement, a wide cylindrical neck 75 is provided for the upper buffer reservoir and the adapter 76 forms a cap mounted on this neck. The cap 76 can thus comprise an annular recess 77 into which the neck 75 fits and is internally threaded at 78 to receive the externally threaded sleeve 79 whose female taper is designed to accommodate the cold finger. A locking ring 80 is threaded onto the sleeve 79 and is provided with a slot 81 which registers with a bore 82 of the cap 76 when the nut 80 is tightened to permit any required appliance (eg. a thermometer) to be inserted into the buflfer reservoir via this bore. Adjustment of the height of the finger 20 with this system is carried out in the previously described manner.

The invention described and illustrated is believed to admit of many modifications within the ability of persons skilled in the art, all such modifications being considered to come within the spirit and scope of the appended claims.

I claim:

1. In an apparatus for gel electrophoresis having an upright gel column, a bulfer reservoir above said gel column, a buffer reservoir above said gel column, a membrane below said gel column defining therewith an elution chamber, an elution tube extending centrally through said column, means for supplying an elution liquid to said chamber around the periphery thereof, and a cooling jacket surrounding said gel column, the improvement which comprises annular collar means connected with said column; and generally tubular membrane-support means adjustably mounted in said collar means and carrying said membrane.

2. The improvent defined in claim 1 wherein said collar means comprises a pair of threadedly interconnected annular collar members, one of said collar members being affixed to said column and the other of said collar members threadedly receiving said membrance-support means.

3. The improvement defined in claim 2, further comprising a hollow base forming a receptacle for an electrically conductive liquid, and means for removably mounting said rcollar means upon said base, said membrane-supporting means comprising a tubular member extending into said base and adapted to be filled with an electrically conductive liquid.

4. The improvement defined in claim 3 wherein said tubular member is closed at its upper extremity by said membrane and at its lower extremity with an electrolyte permeable sheet.

5. The improvement defined in claim 4 wherein said other of said rcollar members is formed with an upwardly extending cylindrical boss receiving said tubular member, further comprising annular sealing means interposed between said tubular member and said other collar member.

6. The improvement defined in claim 5 wherein said annular sealing means includes at least one O-ring received within a groove formed in the outer periphery of said tubular member.

'I. The improvement defined in claim 1 wherein said cooling jacket extends downwardly beyond said column and said membrane-support means projects upwardly into said column and defines with said jacket an annular compartment communicating with said elution chamber.

8. The improvement as defined in claim 1 wherein said collar means and said membrane-support means are composed of a transparent synthetic resin.

9. The improvement defined in claim 1 wherein said means for supplying said elution liquid to said elution chamber includes heat-exchanger means in said jacket for bringing the temperature of said elution liquid to substantially the temperature of said column.

10. The improvement defined in claim 1 wherein said membrane-supporting means is a cylinder, said membrane consituting a rigid microporous disk secured to said cylinder.

11. The improvement defined in claim 10 wherein said disk is composed of glass.

12. The improvement defined in claim 1, further comprising means at said reservoir for adjusting the position of said tube relative to said gel column.

13. The improvement defined in claim 12 wherein said. reservoir is formed with a mouth aligned with said column, said means for adjusting said tube comprising an adapter mounted on said reservoir at said mouth, a sleeve carrying said tube threadedly received in said adapter and vertically adjustable relatively thereto, and a locking nut threaded onto said sleeve for tightening against said adapter to lock said sleeve with respect to said adapter.

14. In an apparatus for gel electrophoresis having an upright gel column, a butter reservoir above said gel column, a membrane below said gel column defining therewith an elution chamber, an elution tube etxending centrally through said column, means for supplying an elution liquid to said chamber around the periphery thereof, and a butter chamber below said membrane, the improvement which comprises adjustable support means for said membrane, the latter being a rigid microporous disk carried by said support means.

7 15. The improvement defined in claim 14 wherein said disk is composed of glass.

16. The improvement defined in claim 14 wherein said support means comprises a tubular member, the interior of said member forming part of said 'bufier chamber.

References Cited UNITED STATES PATENTS 2,535,395 12/1950 Dinsley 204180 3,290,240 12/1966 Neren 204299 3,326,790 6/1967 Bergrahm 2041 80 8 OTHER REFERENCES HOWARD S. \VILLIAMS, Primary Examiner.

JOHN R. MACK, Examiner.

, E. ZAGARELLA, Assistant Examiner.

WW UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 303750187 Dated 1 February 1971 Invent '(s) Joseph BUCHLER It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column l line 40, correct the spelling of with Column 5, lines 2 and 3, read preparative for "perparative' Column 5, line 74 (claim 1, line 3) delete "a buffer reservoir above said gel column,

Column 6, line 13 (claim 2, line 5) read membrane for "membrance" Column 6, lines 17 and 18 (claim 3, lines 4 and 5) read membrane-support for "membrane-supporting" Signed and sealed this L th day of May 1971 (SEAL) Attest:

EDWARD M.FLETCHER,JR. WILLIAM E. SGHUYLER, JR. Attesting Office Commissioner of Patents

Claims

1. IN AN APPARATUS FOR GEL ELECTROPHORESIS HAVING AN UPRIGHT GEL COLUMN, A BUFFER RESERVOIR ABOVE SAID GEL COLUMN, A BUFFER RESERVOIR ABOVE SAID GEL COLUM, A MEMBRANE BELOW SAID GEL COLUMN DEFINING THEREWITH AN ELUTION CHAMBER, AN ELUTION TUBE EXTENDING CENTRALLY THROUGH SAID COLUMN, MEANS FOR SUPPLYING AN ELUTION LIQUID TO SAID CHAMBER AROUND THE PERIPHERY THEREOF, AND A COOLING JACKET SURROUNDING SAID GEL COLUMN, THE IMPROVEMENT WHICH COMPRISES ANNULAR COLLAR MEANS CONNECTED WITH SAID COLUMN; AND GENERALLY TUBULAR MEMBRANE-SUPPORT MEANS ADJUSTABLY MOUNTED IN SAID COLLAR MEANS AND CARRYING SAID MEMBRANE.