CA2086535A1

CA2086535A1 - Device for preparatory electrophoresis

Info

Publication number: CA2086535A1
Application number: CA002086535A
Authority: CA
Inventors: Bertold J. Radola
Original assignee: Individual
Current assignee: Serva Feinbiochemica GmbH and Co
Priority date: 1990-07-07
Filing date: 1991-06-29
Publication date: 1992-01-08
Also published as: JPH05508577A; EP0539399A1; DE4021728A1; WO1992000795A1

Abstract

Abstract The invention relates to an electrophoresis chamber of modular construction having a dimensionally independent capillary cooling system.

Description

S012611J.39 3/348-PCT/Dr.Kl/A-5389t Apparatus for pre~arative electrophoresis The invention relates to an apparatus for preparative electrophoresis, and more particularly a dimensionally independent cooliny system for this apparatus.

Electrophoresis is currently the most efficient analytical method for separating proteins. There are also numerous techniques of preparative electrophoresis, but these are used predominantly for separation on a scale of milligram quantities of proteins. A major problem in scaling up is the removal of the Joulean heat produced during the passage of the current. A
particularly successful technique is preparative isoelectric focussing in layers oE granulated gels, by means of which gram quantities of proteins can be separated with high resolution (Radola, B.J., Methods Enzymol. 1984, 104, 256-275). In this system of separation, the layer thickness is limited to about l cm and moreover the separating distance cannot be extended, which means that the separating volume can only be increased by varying the width of the layer. However, such widening of the scale is subject to strict practical limits. Other preparative systems known from the literature having cylindrical geometry are equally unable to be scaled up with either radial or axial cooling (Rilbe, H. and Petterson, S., in: Arbuthnott, ~-~
J.P. and Beeley, J.A. Isoelectric Focusing, Butterworth, London 1975, pp. 44 - 57). The aim of the present invention is to provide an apparatus for preparative electrophoresis which can be used to separate larger quantities of substance.

According to the invention, an apparatus for preparati~e electrophoresis is proposed which is characterised by the modular construction of the electrolyte chamber from a plurality of individual compartments, the con~partments being connected by partition elements with membrane-like properties, each compartment containing a cooling element which consists of parallel capillaries.
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An essential part of each compartment - which contains -the electrolyte - is a cooling element made up of capillaries, in which thin capillaries are arranged parallel to one another, so that their spacing from one another and from the boundary surfaces of the compartment does not amount to more than a few millimetres at any point. The boundary surface of the compartment is formed by the adjoining element (also re~erred to as the partition element) with the membrane~
like properties. Generally, the spacing of the capillaries from one another is between 3 and 10 mm, preferably between 5 and 7 mm. The spacing from the ~;
nearest adjoining surface - the partition element between two compartments - should be not more than 5, preferably not more than between :L and 3 mm, the spacing being measured from the surface of the capillary outwards. T~e diameter of the capillaries should be as small as possible, although there are technical limits imposed because of the material properties and the internal diameter necessary to maintain an effective flow of cooling fluid~ Generally, the outer diameter of the capillaries is between 1 and 3 mm.
:- ' An important requirement of the capillaries is that they -should not be electrically conductive and should be neutral and resistant to the chemicals used (electrolyte, protein, etc.).

Suitable materials from which capillaries may consist .; .;

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include, for e~ample, metals, provided that they are coated on the outside with electrical non-conductors, e.g. with plastics (polyethylene, Teflon etc.), plastics and glass.
-The cooling of the capillaries is a function of thelength, wall thickness and hPat conductivity of the material and the throughflow rate and efficiency of the cooling units. The cooling principle according to the invention may be used up to a total volume of at least 100 litres or more and is determined primarily by the length of the separating distance and the separating time required. Separating times of more than 24 hours appear to be justified only in especially problematic situations. The cooling efficiency has been tested in compartments with different arrangements of capillaries (spacing, length, diameter, material, throughflow rate of cooling fluid, depth of layer). Temperature measurements at different loads of the system have shown that the temperature is constant throughout the entire system, even at critical points, e.g. near the electrodes, up to a load of 0.3-0 4 watts/cm. This watt load is many times greate~ than the development of heat which can be expected during electrophoresis.

The capillary cooling system according to the invention is also referred to as a dimensionally independent cooling system, since it automatically adapts to the volume of the electrolyte chamber because of the modular construction of said chamber.
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compartment with the cooling element made up of ~ " -capillaries forms a constructional unit, the compartment -having the following constructional features: opposing ~ ~`
side portions form together with a base portion a flat ~;-frame which is generally open at the top. The height of j ;

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- 4 - ~-the side portions and the length of the ~ase portion may be of any desired dimensions within a wide range,;-~ -thereby defining the height and width of the internal ;
dimensions of the electrophoresis chamber. For example, the height may be 30 cm and the width 40 cm, the external dimensions being greater depending on the ~-materials used. Preferred materials are chemically --;~
resistant plastics which are mechanically easy to work, such as Plexiglas and polyethylene. The edges of the ~ -opposing side portions, together with the edges of the base portion, form a surface adjacent to which is a partition element. The spacing of these two surfaces from each other, i.e. the thickness of the side portions and of the base portion, is prescribed by the cooling efficiency of the capillary cooling system. As a rule :
the spacing will be 2 to 10 mm, preferably 2 to 5 mm.
The number of compartments defines the separating efficiency of the electrophoresis chamber. For a given constant volume of the chamber, a smaller thickness of the compartment means a larger number of compartments and, at the same time, an improved separating efficiency of the system. Thus it is desirable to obtain a compartment of reduced thickness, but constructional limits are imposed on this by the capillary cooling system. -:
In one particular embodiment the base portion has an increasing thickness so as to produce a gradient inside the compartment so that the electrolyte solution -optionally together with the separated protein - can be completely drained away through an outlet opening provided at the lowest point.
''' ~ ' Should the compartment and the cooling element formed from the capillaries form a constructional unit (Fig. 2), each side portion contains one or more recesses (openings). As a result of the modular : .

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construction consisting of a plurality of compartments, these recesses form cooling water channels through which the ~ooling liquid flows in at one end - through the capillaries - and flows out of the opposing cooling water channel. It does without saying that the electrophoresis chamber according to the invention, consisting of individual compartments, contains connectors so that the cooling water channels can be suppli~d with cooling liquid.

If it is desired or necessary to increase the cooling efficiency, the capillaries of each compartment, either on their own or assembled in blocks, may be separately supplied with cooling fluid. The temperature of the cooling fluid may be adapted to the particular problem of separation arising - e.g. using cryostats - e.g. in a temperature range between 1 to 30C or for low temperature electrophoresis at between -10 and -30C.
For separation in the presence of high polyol concentrations the preferred temperature range is around 20C.
.: ' The capillaries are connected to the side portions so as to make contact with the cooling water channel, but no cooling fluid-is able to penetrate into the interior of `
the electrophoresis chamber.
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sealed off from one another by a partition element.
Accordingly, the edges of the side portions and the ~ -edges of the base portion of each compartment are shaped so as to perform a sealing function and to prevent any electrolyte liquid from escaping. They may, for example, contain sealing profiles made of rubber or ~
another suitahle material such as Teflon or silicone. ~-:
The partition elements serve to prevent the exchange of ~
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fluids between adjacent compartments, whilst the proteins to be separated are able to diffuse, i.e. the partition elements act as a membrane. Any substances -~
which have this membrane function and are also sufficiently mechanically stable can be used. Suitable materials include, for example, porous polymer films, ceramic membranes, or industrial fabrics coated with a very thin gel. Such fabrics are described for example in German Offenlegungsschrift 37 36 087, the contents of which are referred to here.

Particularly preferred materials are ultra-thin fabric-sup~orted polyacrylamide gels or agarose gels with a thickness of about 50 ~m to 2 mm, preferably 50 to 100 ~m. Thin fabric-supported gels of this kind may be placed directly between two compartments without the need for any other constructional features for receiving a partition element. The gels are fixed between the compartments by the pressure exerted by an external clamping device. The surface area of the gels is of such dimensions that the cooling water channels are not covered.

In one embodiment of the invention it is provided that the partition-elements consist of thin frames with a ~ ;
fixedly connected fabric on which the gel can be polymerised. This embodiment enables rapid construction of the electrophoresis chamber and easier replacement of the partition elements. It goes without saying that in ;
this case the frames must have the same sized recesses for the cooling water channels as the associated compartments.

It is possible that the gels may contain additives such as are normally used in electrophoresis. In this way it is possible to adapt the electrophoresis chamber according to the invention to the variety of separating - . , .. : . . - ............ ., . - . . . : . .: : ,-' ' ' ' : . . .; " ' : ,'' -- 7 - 2~ 3~
problems posed. Thus, for example, when polyacryamide gels are used, additional ~unctional groups may be included in the gel, as is already known in the technique of isoelectric focu~sing in immobilised pH
gradients.

The dimension-less capillary cooling system according to the invention may take various forms. The preferred embodiment, in which the capillaries are fixedly connected to the side portions of the compartments and are supplied with ~ooling liquid through cooling water channels, has already been described above.
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In another embodiment the capillaries of each compartment are joined together to form an "endless"
capillary and are attached to a cooling liquid system.
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In another embodiment the capillary cooling system is not fixedly connected to the compartment. The ~ -capillaries àre arranged in a flat frame which also contains the apparatus for supplying and removing the -cooling liquid. The dimensions of the frame are such that it can be inserted in grooves provided in the compartment. It is necessary that the frame with the capillaries be arranged parallel to the boundary surfaces of the compartment. ~ ~
~ : "' ' :' In the first and last compartments are the electrodes (cathode and anode) of the electrophoresis chamber.
Although the construction of the electrodes may vary within the scope of conventional forms, it has proved advantageous to use an electrode constructed like a net.
A "coarse-mesh" net (mesh size 1 to 3 mm) made of an ;
inert material (e.g. plastics) has a conductive material, preferably a platinum wire, meandering through it. It is also suitable to use nets which are woven exclusively from a platinum or platinum/iridium wire.

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Graphite electrodes or titanium/platinum electrodes are also suitable. The surface area of the electrode corresponds substantially to the inner surface area of the compartments. Because of the high field intensity between 50 ml and 200 Volts/cm and the consequent development of gas in the electrode space it is advantageous for the two compartments containing the two electrodes to have a substantially larger volume than the other compartments. This avoids excessive foaming.
The segmented construction of the electrophoresis chamber according to the invention allows the -electrolyte solution of the compartments with the electrodes to have a different composition from the other compartments. Thus, for example, a higher content of a polyol can reduce the foaming in the region of the electrodes.

The electrophoresis chamber according to the invention -is made up of a number of sections, with alternating compartments and partition elements. The two outer compartments contain the two electrodes. This electrophoresis chamber, with its layered construction, is held together with a clamping device in order to seal the individual components (compartments and partition elements) so-that no electrolyte solution can escape outwards and no exchange of liquids can take place between adjacent compartments. -With the electrophoresis chamber according to the invention it has proved advantageous, for the purpose of anticonvective stabilisation of the separated substances, to fill the chamber with a 40 to 80 solution of a polyol, e.g. glycerol, saccharose, sorbitol or a mixture of these polyols. The individual compartments are separated from one another by fabric-supported polyacrylamide gels. With this anticonvective stabilisation it is possible to ta]ce samples from all '' ' ': .

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'2~6~35 the segments during the separation process and, after separation, to elute the proteins easily without any disruptive mixing. During the introduction of isoelectric focussing in pH gradients stabilised by polyol density gradients, it was found that high polyol concentrations are compatible with the electrophoresis process.

In particular, when the chamber has a high load of substance, it may be necessary constantly to circulate the contents of the individual compartments by means of ~ -a multichannel pump in order to prevent -~
electrodecantation in this manner. Electrodecantation would cause the separated substances to accumulate in the lower part of the compartments, which would interfere with separation and would therefore be ;
undesirable. For optimum separation it is desirable for the separated substances and electrolytes to be -distributed uniformly over the entire cross-section of the compartments. The pump is excluded by means of the outflow channel of the individual compartments and the liquid pumped round is recycled through a tube into the upper part of the particular compartments.

Compartmentalisation with fabric supported polyacrylamide gels has a number of advantages.
Polyacrylamide gels are a matrix best known from analytical experiments which does not interfere with isoelectric focussing and other electrophoretic separation processes. The layer thickness of the fabric ~ -supported by acrylamide gels~may be selected anywhere between 0.05 and 2 mm. In this way it is possible to make the ratio of the liquid phase to the gel phase in ~ ;
the separation chamber variable, which may have a crucial effect on the resolution. The fabric-supported gels are mechanically stable and allow good compartmentalisation. The fabric-supported gels can be 2~8~35 -- 10 -- ~
washed, dried and rehydrated. The composition of the fabric-supported gels may be as desired, within specific degrees of cross-linking.

The use of polyacrylamide gels also makes it possible to introduce additional functional groups into the gel, as is known from the technique of isoelectric focussing in immobilised pH gradients (Gorg, A., Fawcett, J.S. and Chrambach, A, Adv. Electrophoresis 1988, 2, 1-43).

The compartments may contain sensors for measuring ;~
important parameters, such as the pH, temperature, UV, IR, measuring the activity of radiolabelled samples, conductivity, etc. The electrophoresis chamber according to the invention can be fully automated for discontinuous operation if additionally an automatic sample recorder and sampling device is installed.

Usually, the electrophoresis chamber according to the invention is operated in a horizontal position. If, however, the frames of the compartments are closed on all sides - with the exception of an opening for filling and emptying the compartment - the electrophoresis chamber can also be operated vertically.
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In order to separate the protein, for example, the following procedure is used: -Of the compartments filled with electrolyte solution, ~-one or more compartments are emptied and filled with a mixture of the sample to be separated and electrolyte solution. The progress of separation can be monitored -either by direct sampling from the individual compartments or, if the compartments contain suitable sensors, by means of the data obtained by measurement.
The separated samples are isolated by simply emptying ;
the relevant compartments.

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During isoelectric focussing, all the compartments can be emptied, to be filled with sample solution. In the electric field the sample is then separated in - -accordance with the isoelectric points o* the individual components.

Fractionation of carrier ampholytes is an important stage in the production of carrier ampholytes for isoelectric focussing. With a high-resolution separating chamber it should be possible to prepare i~
narrow pH ranges of carrier ampholytes, with better defined properties than was possiblè with the processes used hitherto. Narrow pH ranges of carrier ampholytes are important for separations in which a high resolution is required, as is the case, for example, when investigating genetic markers.

In a number of experiments, mixtures for synthesising the carrier ampholytes were fractionated and the isolated fractions were tested in analytical focussing -tests. Thanks to the excellent cooling efficiency of the new separation chamber, the concentrated synthesising mixture (35%) could be fractionated directly, i.e. without dilution, thereby saving the expensive concentration of the dilute fractionated carrier ampholyte which was hitherto required. After a separating time of 44 hours the pH gradient showed a substantially linear course, with a minimum conductivity characteristic of this pH range at a neutral pH. During analytical refocussing the pH gradients measured in the gel coincide with the pH range isolated in the preparative experiment, with in some cases excellent ~ -linearity over a narrow pH range (e.g. pH range 3 to 4).

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The fractionation of carrier ampholytes shows that it is possible to carry out separations in the new separating chamber even when the sample or the bu~fer electrolyte has a high conductivity. Such separations might also be of interest in the case of other low molecular substances.

For separating various proteins, the pH gradient and the Vh product must be optimised. This presupposes a stability of the p~ gradient in the electrical field.
In an experiment in pH 4 to 9 Servalyt carrier ampholyte ~ -~
the pH gradient was constant for Vh products of 6000 to 13000 Vh. In preparative refocussing of narrow pH
ranges, the pH range coincided with the range originally -isolated. This experiment shows that cascade focussing in two or more steps is possible, which could significantly improve the resolution for components which are difificult to separate. Figure 5 shows the stability of the pH gradient during preparative isoelectric focussing with differ~ent Vh products.
Experiment: Separation of 3 mg/ml albumin protein in 1000 ml pH 4 to 9 T (1%) and 40% glycerol.

As shown in Figure 1, the electrophoresis chamber according to ~he invention is characterised by its --modular construction. The compartments (2) are arranged one after the other on a base plate (l); this drawing does not show the partition elements located between two compartments. Adjoining the compartments which contain the electrodes (3) are two stable end-blocks (4) which have means (5) for receiving two guide rails (6). The ~lates, the guide rails and the end portion (8) enable the individual compartments to be fixed, so as to form a fluidtight electrophoresis chamber. If the guide rails are in the form o~ a round rod or a ~ube, they may contain a thread.
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The end portion (8) is fitted on and screwed tight by means of the nuts, so that the necessary pressure is exerted on the electrophoresis chamber via the end portion movably mounted on the guide rails. The end-blocks ~4)~ simultaneously serve to seal off the recesses (7) for the two coolant channels so that the cooling fluid cannot escape. Similarly, the end blocks contain means (9) for connecting the capillary cooling system (lI) to a cryostat or another supply of cooling fluid (not shown in the drawings). Seals (12) between the ~ ~
compartments (2) prevent the liquid escaping. The -recesses (7) form two opposing coolant channels. The electrical connections to the electrodes are not shown either. of course, the compartments may be held together by any corresponding, technically equivalent means.

With one exception, the compartments are shown only in front elevation, to make the construction of the electrophoresis chamber clearer.
' Generally, the chamber consists of at least 5 ;
compartments; a smaller number would be possible, but would only be practical in except:ional cases, because of the separating performance. Shortened separation routes with 5 compartments or less are reasonable if they are used in so-called cascades. Preliminary separation of the sample occurs in a first eIectrophoresis chamber, then the contents of a compartment are separated into further fractions in another chamber. -~ ;

This procedure may be repeated several times.
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The combination of several electrophoresis chambers with a small number of compartments to form a cascade enables ~-~
rapid separation of, e.g., a protein mixture.

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Fig. 2 is a section through a compartment (2) with --capillary cooling system fixed therein. Two side parts (14) and a base part (15) are joined to form a frame (16). The base part is of varying thickness and at the lowest point there is a closable drainage channel (17) above which a multi-channel pump can be attached in order to circulate the contents of the individual compartments to prevent electrodecantation. Openings (7) are provided in the side parts. The coolant channels are thus formed by combining several frame , parts. The capillaries (18) are fixedly connected to ~-, the side parts (14).

Fig. 3 is a section through a compartment (2) with subdivided openings (7a) and (7b) for forming separate , coolant channels. For more effective cooling, cooling fluid may be passed along them in counter-current flow, '~
for example. Fig. 4 shows a separating element (19) ':, with a fabric-supported gel (20) incorporated therein.
The side parts (21) contain resses (22) for forming coolant channels. The side parts (21), the base part (25) and an upper part (23) form a solid frame (24) supporting the fabric on which the gel is polymerised.

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Claims

1. Apparatus for preparative electrophoresis with an electrolyte chamber of modular construction consisting of a plurality of compartments, the individual compartments being separated from one another by elements (partition elements) having membrane-like properties, characterised in that each compartment contains a cooling element consisting of parallel capillaries, the spacing of the capillaries from one another being up to 10 mm, preferably up to 5 mm, and the spacing from the boundary surfaces of the compartments being not more than 5 mm.

2. Apparatus according to claim 1, characterised in that the partition element consists of a polymer film having membrane-like properties.

3. Apparatus according to claim 1, characterised in that the partition element consists of a fabric-supported polyacrylamide or agarose gel.

4. Apparatus according to claim 1, 2 or 3, characterised in that the capillaries consist of steel and are coated with an electrically non-conductive material.

5. Apparatus according to claim 1, 2, 3 or 4, characterised in that the capillaries consist of plastics.

6. Apparatus according to claim 1, characterised in that it consists of a) at least 5 compartments, provided with parallel cooling capillaries at a small spacing from one another -, b) two compartments for receiving the electrodes, c) means for supplying the capillaries with cooling fluid, and d) means for holding the compartments together.

7. Apparatus according to one of claims 1 to 6, characterised in that it contains means for mixing the electrolyte in the individual compartments in order to prevent electrodecantation.

8. Apparatus according to claim 6, characterised in that it contains a multichannel pump for mixing the electrolyte in the individual compartments in order to prevent electrodecantation in the individual compartments.

9. Dimensionally independent cooling system for preparative electrophoresis, characterised in that it consists of a-flat frame which also contains a device for supplying cooling fluid, having parallel cooling capillaries of a small external diameter of between 1 and 3 mm, arranged at a small spacing of between 3 and 10 mm from one another.

10. Cooling system according to claim 9, characterised in that the capillaries consist of steel and are coated with an electrically non-conductive material.

11. Cooling system according to claim 9, characterised in that the capillaries consist of plastics.