GB2144768A - Gel electrophoresis - Google Patents

Abstract

An apparatus for performing gel electrophoresis comprises two tanks located one above the other and a wall 13 separating the two tanks, each tank having a compartment located centrally within it and each compartment housing an electrode 17,22, wherein the wall dividing the two tanks has circumferentially spaced through-holes each capable of receiving a sample of gel (in recipient 8) that is to be subjected to electrophoresis, and wherein each compartment has formed in the wall thereof apertures 21,26 facing the holes in the wall dividing the tank. The electrodes may comprise an electrode spool shaft 20,25 and spirals of platinum wire fixed in grooves on the electrode spool shaft. <IMAGE>

Description

SPECIFICATION Apparatus for electrophoresis with polyacrylamide-gels with an oriented current field The invention relates to an apparatus for the electrophoresis with polyacrylamide-gel with an oriented current field, by the aid of which zonedistortions can be practically eliminated, serial examination with a routine character can be performed reproducibly and quickly, the requirement of chemicals is low and which can be operated with the simultaneous elimination of electric shockarising generally due to electric voltage used for measuring wet media in compliance with the prescriptions of shock-proofness.

From technical literature dealing with gas-electro-phoresis with polyacrylamide, it is well known that with one type of the so-called zone-electrophoretic processes, synthetic polyacrylamide-gel is used as electrophoretic carrier. Compared to earlier applied electrophoretic processes the main advantage of the zone-electrophoretic processes lies in the increased resolving power and this is most characteristic for the electrophoresis with polyacrylamide-gel. In this case namely in addition to the separation of the charged macromolecules (e.g.

proteins, nucleic acids, etc.) according to net electric charge the so-called molecule filtering effect also arises. The essence of this lies in the separa tion of ions according to shape and size. Molecule filtering effect is characterizing for electrophoresis with polyacrylamide-gel, since separation of ions according to shape and size, respectively, is preferred to the separation according to charge. Molecules are filtered with the highest efficiency within the range of molecular weight between 104 and 107, while its extent can be controlled by the change of average pore size in dependence of the material concentration of the gels.

Resolving power of the method can be further increased by the application of the so-called discontinuous buffer systems. In this case the gelbuffer shows properties that are different from those of the electro-buffer, and also, with systems of this kind in general the gel itself consists of several layers with different concentrations and pHvalues. By the application of discontinuous buffer systems an electrochemical effect appears, which prevails through the high-grade concentration of the components of the sample-solution and the formation of extremely thin starting zones. As a result, a most efficient separation of the components of macro molecular mixtures can be achieved.

Owing to the most advantageous features the method has been widely used. The main possiblities of application are the separation of the components of peptides, proteins, nucleotides, nucleic acids, as well as different cell fragments (e.g. diaphragms, ribosomes). In practical life the method has been applied in particular in laboratory diagnostics,in clinics and for testing foodstuffs.

Owing to their considerable importance in practice, in the technical literature a number of apparatuses are described, and several apparatus types are available in commerce.

For example, simple gel-electrophoretic apparatuses are described in the publications Ann. N.Y.

Acad. Sci. 121, 404 (1964) and Ann, N.Y. Acad. Sci.

121, 428 (1964), as well as the volume "Electrophoresis of Proteins in Polyacrylamide and Starch Gels" of the series "Laboratory Techniques in Biochemistry and Molecular Biology" (Authors: T.S.

Work and E. Work) page 48 (Published: North Holland, 1972).

The members of the apparatus-family produced and marketed by Savants Instruments Inc. made of plexiglass and vertically arranged, of the type DEC, for electrophoresis with polyacrylamide-gel, are operated with gel containing glass pipes with a length of 10-20 cm. and with an inner diameter of 6 mm. The inner diameter of the cylindrical buffer tanks amounts to 15 cm., their height to 18-25 cm., accordingly their complete buffer requirement is about 3 to 4.4 litres; the net weight of the apparatus equals to about 1.3 to 1.7 kg.

The vertically arranged apparatus for electrophoresis with polyacrylamide-gel, marketed by Pharmacia Fine Chemicals and made of polycarbonate of the type GE-4 is operated with gel-containing pipes with an inner diameter of 2.7 or 5 mm., of which in total 16 pieces are needed. The volume of the square buffer tanks is: the upper tank about 0.4 litres, the lower tank about 2 litres.

The gel-electrophoretic cells of the models 150A and 151 of Bio-Rad-Laboratories operate with pipelengths between 150 mm. and 300 mm., accordingly these are large dimensioned-apparatuses requiring a considerable quantity of electrode buffer.

The apparatuses partly do not meet the requirements for safe work performance due to their too simple design, i.e. they do not guarantee proper reproducibility of the tests in the course of serial examinations of routine character, partly becausedue to their over complicated design-their use is wearisome; also the dimensions are too large and the required chemicals for each serial test is considerable.

With all the known apparatuses the leads of the electrodes result in an asymmetrical arrangement of the electrodes, increasing the extent of zone-distortion. Zone-distortion manifests itself in the mutual merging of the components to be separated.

This phenomenon makes the evaluation and recognition of thin zones difficult, and in some cases impossible.

In the course of electric measurings performed on wet media or separating processes, increased attention is to be paid to the strict instructions of contact protection. The fundamental disadvantage of known solutions lies in that they do not yield the proper protection against electric shock.

Abroad and in Hungary several persons have suffered badly from injuries while operating gel electrophoretic apparatuses.

The aim of the invention is to eliminate said deficiencies. We wish to develop a gel-electrophoretic apparatus, by the aid of which thin zones can be recognised without merging, evaluation thereof be comes possible; also due to the small dimensions the apparatus can be easily manipulated, velocity of separation can be increased, serial tests require a small quantity of chemicals, at the same time requirements relating to contact protection are mostly met.

The invention is based on the recognition that by attenuating the current components exerting an adverse effect on ion mobility in the buffer solutions used in gel-electrophoresis and by the proper orientation of the current, zone-distortions can be practically eliminated.

In accordance with the invention, the aim set on basis of the aforementioned recognition has been achieved with an apparatus for the electrophoresis with polyacrylamide-gel, with which in cylindrical lower and upper buffer tanks, respectively the lower and upper electrodes are arranged and the gel-containing recipients discharge into the buffer tanks. In every buffer tank an electrode carrying cylinder is arranged, coaxially with the symmetry axis of the tank and in every electrode carrying cylinder there is a centralized electrode-spool shaft to be found. The electrodes are spirals wound from a platinum :vire, which are fixed in grooves formed in the electrode-spool shafts.On the wall of the single electrode carrying cylinders, on the part lying nearest to the electrodes, holes are formed facing the gel-recipients, through which the buffer solution can flow to the electrodes: independently of the level of the buffer solution the electrodes are submerged in the buffer solution always up to the same level. The gel-recipients are expediently fixed in the base of the upper buffer tank so that these should lie at an equal distance from the symmetry axis of the disc and the distance from each other should be also identical. Sealing can be achieved by sealing rings made of silicona rubber.

In order to meet the strict requirements relating to contact protection, current input connections of the electrodes are formed so that by closing and opening, respectively of the covering disc current supply is started or stopped. This can be achieved in such a manner that the upper electrode is connected electrically with a plug which is fixed to the upper covering plate of the upper electrode carrying cylinder, while the lower electrode is electrically connected to a plug fixed to the outer wall of the lower buffer tank, whereby the connecting sleeve of the upper plug is connected directly, the connecting sleeve of the lower plug is connected indirectly, through the shank of the sleeve holder to the covering disc of the upper buffer tank.

The invention will be described in detail, by way of an example, with reference to the accompanying drawing illustrating a possible embodiment of an apparatus for electrophoresis with polyacrylamidegel, in a sectional view.

As is obvious from the figure, the apparatus consists of two vertically arranged buffer tanks with circular cross-sections, which are joined to each other by the grooves milled into a base-disc 13 of the buffer tank. The upper buffer tank consists of the base-disc 13 and the wall 5 of the tank, while the lower buffer tank comprises a base-disc 15 and the wall 7. In the centre of the base-discs 13 and 15, electrode spool shafts 20 and 25 are fixed, which are enclosed by electrode carrying cylinders 12 and 14, which are fixed into a circular groove formed at a distance of approx. 1 cm. from the centre. In grooves on the electrode spool shafts 20, 25 electrodes 17 and 22 wound helically from a platinum wire are fixed.The electrode 17 is connected to a plug 16 which is fixed to an upper covering plate of the electrode-carrying plate of the electrode carrying cylinder, while the electrode 22 is connected via the electrode lead 22 electrically with the plug 9 which is fixed to the base-disc 15 outside the buffer space. For receiving the electrode-lead 23 the base-disc 15 is provided with a bore 11. In the base-disc 13, which has a radius of about 5 cm., circumferentially spaced through-bores can be found, in which the gel-recipients 8 (only one shown) are fixed with the seals 6 formed of the silicone rubber rings. The gel-recipients 8 are glass pipes open at both ends, which contain the gel. On the wall of the electrode carrying cylinders 12, 14 in the region of the electrodes 17, 22 there are the holes 21, 26 facing the gel-recipients.

The upper buffer tank is closed by a covering disc 3 provided with a cover-grip 1; the shank of the plug sleeve is vertically attached to the edge of the covering disc 3. The lower end of the shank is provided with a connecting sleeve 10 receiving the plug, which is connected to the outer current generator 24. The sleeve 2 receiving the plug 16 is arranged coaxially with the covering disc 3 and is connected to an external current generator via the current supply lead 19.

The apparatus according to the invention is operated so that first of all the polyacrylamide-gels are prepared in the gel-recipients, the recipients are fixed by means of the sealing 6 in the bores of the base-disc 13, thereafter 200 ml. buffer solution are filled into the lower buffer tank. Now the upper buffer tank is placed on the lower, and 150 ml.

buffer solution are filled thereto. Next the covering disc 3 is placed onto the upper buffer tank in such a manner that the plug 16 should be fitted into the connecting sleeve 2 and the plug 9 into the connecting sleeve 10. Thereafter the current supply leads 19 and 24 are connected to the stabilized current generator.

In the course of separation, current flows from the electrode 17 through the intermediate buffer solution conducting current via the hole 21 and so a quasi focussed current arrives at the polyacrylamide-gel. From the gel, the current also flows through the buffer solution and the hole 26, which exerts a grid-effect, to the electrode 22. Experimentally it can be demonstrated that our apparatus is able to separate zones of about 1 mm. thickness.

The boundary of the zones is sharp, it is free of merging, accordingly, compared to all the known solutions, the apparatus according to the invention is able to give far more accurate results.

The application of the apparatus according to the invention ensures the surprising advantage, in so far as the buffer requirement of a gel-electrophoretic analysis each is at least by one order of mag nitude lower, than with any other apparatuses available in commerce. By using the apparatus according to the invention reproducible results can be obtained in such a manner that by closing the covering disc 3, the parts under voltage cannot be touched, accordingly, the danger of electric shock is excluded with the highest safety.

Claims (8)

1. Apparatus for the electrophoresis with polyacrylamide, with an oriented current-field, with lower and upper cylindrical buffer tanks, as well as with gel-recipients discharging in said buffer tanks, characterized in that in every buffer tank an electrode carrying cylinder (12, 14) is arranged co-axially with the symmetry axis of the tank and in every electrode carrying cylinder (12, 14) there is a centralized electrode spool shaft (20, 25) each to be found, the electrodes are spirals wound from a platine wire which are fixed in the grooves on the electrode spool shaft (20, 25), furtheron on the mantle of each electrode carrying cylinder (12, 14) in the environment of the electrodes (17, 22) holes (21, 26) facing the gel-recipients (8) are formed.

2. Apparatus according to claim 1, characterized in that the gel-recipients (8) are fixed in the base disc (13) of the upper buffer tank with identical radius and distance from each other.

3. Apparatus as claimed in claim 1 or 2, characterized in that the upper electrode (17) is connected electrically to a plug which is fixed to the upper lid of the upper electrode carrying cylinder (12), while the lower electrode (22) is electrically connected with the plug (9), which is fixed to the outer wall of the lower buffer tank, whereby the connecting sleeve (2) of the upper plug (16) is connected directly, the connecting sleeve (10) of the lower plug (9) indirectly, through the shank (4) of the sleeveholder to the upper covering disc (3) of the upper buffer tank.

4. An apparatus for performing gel electrophoresis, which apparatus comprises two tanks located one above the other and a wall separating the two tanks, each tank having a compartment located centrally within it and each compartment housing an electrode, wherein the wall dividing the two tanks has circumferentially spaced through-holes each capable of receiving a sample of gel that is to be subjected to electrophoresis, and wherein each compartment has formed in the wall thereof apertures facing the holes in the wall dividing the tank.

5. An apparatus according to claim 4, wherein all the holes in the wall dividing the two tanks are equally spaced from the electrode in each of the two tanks.

6. An apparatus according to claim 4 or claim 5, wherein the electrode in the upper tank is connected by way of an electrical plug and socket connection to an external lead, one of the plug and socket being formed on a lid closing the upper tank, the other of the plug and socket being secured to the compartment wall in the upper tank.

7. An apparatus according to any one of claims 4 to 5, wherein the top tank is closed by a lid and the electrodes in the two tanks are each connected to external electrical connectors by way of respective plug and socket connectors, wherein one of the plug and the socket of each connector is secured directly or indirectly to the lid so that, when the lid is removed, the electrical connectors are disrupted.

8. An apparatus substantially as hereinbefore described in connection with and as illustrated in the accompanying drawing.