WO1992000795A1

WO1992000795A1 - Device for preparatory electrophoresis

Info

Publication number: WO1992000795A1
Application number: PCT/EP1991/001219
Authority: WO
Inventors: Bertold Radola
Original assignee: Serva Feinbiochemica Gmbh & Co.
Priority date: 1990-07-07
Filing date: 1991-06-29
Publication date: 1992-01-23
Also published as: JPH05508577A; CA2086535A1; EP0539399A1; DE4021728A1

Abstract

An electrophoresis chamber of modular design with capillary cooling system non-dependent on chamber size.

Description

Device for preparative electrophoresis

The invention relates to a device for preparative electrophoresis and in particular to a dimension-independent cooling system for this device.

Electrophoresis is currently the most powerful analytical method for protein separation. Numerous techniques of preparative electrophoresis are also known, but these are mainly used for separations on a scale of milligram amounts of proteins. A major problem with scale up ("scale up ^M ) is the dissipation of the Joule's heat generated during the passage of current. A particularly successful technique is the preparative isoelectric focusing in layers of granulated gels, with the aid of which gram amounts of proteins are separated with high resolution could (Radola, BJ, Methods Enzymol. 1984, 104, 256-275).

In this separation system, the layer thickness is limited to approximately 1 cm, and the separation distance cannot be extended either, so that the separation volume can only be increased by varying the width of the layer. Such an extension of the scale has narrow practical limits. Also other preparative systems with cylindrical geometry known from the literature can neither be transferred to a larger scale with radial or axial cooling (Rilbe, H. and Petterson, S., in: Arbuthnott, JP and Beeley, JA Isoelectric Focusing, Butterworth, London 1975, pp. 44-57). It is the object of the present invention to provide a device for preparative electrophoresis which enables the separation of larger amounts of substance.

REPLACEMENT BLADE According to the invention, a device for preparative electrophoresis is proposed, which is characterized by a modular structure of the electrolyte chamber from a large number of individual compartments, the compartments being linked by separating elements with membrane-like properties, and each compartment containing a cooling element consisting of capillaries arranged in parallel.

An essential component of every compartment - which contains the electrolyte - is a cooling element made up of capillaries, in which thin capillaries are arranged parallel to each other, so that the distance to each other and to the boundary surfaces of the compartment is never more than a few millimeters. The boundary surface of the compartment is formed by the adjacent element (also referred to as a separating element) with the membrane-like properties. The distance between the capillaries is generally between 3 and 10 mm, preferably between 5 and 7 mm. The distance to the next adjacent surface - the separating element between two compartments - should not be more than 5, preferably not more than between 1 and 3 mm, the distance being measured from the surface of the capillary. The diameter of the capillaries should be as small as possible, but due to the

Material properties and an internal diameter - necessary for maintaining an effective coolant transport - are technical limits. In general, the outer diameter of the capillaries is between 1 and 3 mm.

An important requirement for the properties of the capillaries is that they are not electrically conductive

REPLACEMENTB and behave neutrally and consistently towards the chemicals used (electrolyte, protein etc.).

Suitable materials from which capillaries can consist are, for example, 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 the length,

Wall thickness, thermal conductivity of the material that

Flow rate and performance of the

Cooling units. The cooling principle according to the invention can be used up to a total volume of at least 100 liters and more and is primarily determined by the length of the separation section and the separation time to be used with it. Separation times of over

24 hours only appear for special ones

Problems justified. The cooling capacity was measured in compartments with different arrangement of the capillaries (distance, length, diameter, material,

Flow rate of the coolant,

Layer height) checked. Temperature measurements at different loads on the system have shown that the temperature in the whole system, even at critical ones

Places e.g. near electrodes, up to one

₃ load of 0.3-0.4 watts / cm is constant. This

Wattage exceeds many times that of

Expected heat development by electrophoresis.

The capillary cooling system according to the invention is also referred to as a dimension-independent cooling system, since it is automatically adapted to the volume of the chamber due to the modular construction of the electrolyte chamber.

REPLACEMENT LEAF In one embodiment according to the invention, a compartment forms a structural unit with the cooling element made up of capillaries, the compartment having the following structural features: Opposite side parts together with a base part form a flat frame which is generally open at the top. The height of the side parts and the length of the bottom part can be chosen in a wide range, whereby the height and width of the internal dimensions of the electrophoresis chamber are defined. For example, the height is 30 cm and the width is 40 cm, with the outside dimensions being larger depending on the materials used. Preferred materials are chemically resistant plastics that can be processed mechanically, such as plexiglass and polyethylene. The edges of the opposite side parts together with the edges of the bottom part each form a surface, which is followed by a separating element. The distance between these two surfaces, ie the thickness of the side parts and that of the bottom part, is predetermined by the cooling capacity of the capillary cooling system. As a rule, the distance is 2 to 10 mm, preferably 2 to 5 mm. The number of compartments defines the separation performance of the electrophoresis chamber. For a given equal volume of the chamber, a smaller thickness of the compartment means a larger number of compartments and thus at the same time an improved separation performance of the system. A compartment of small thickness should therefore be aimed at, but the capillary cooling system places structural limits on this.

In a special embodiment, the bottom part has an increasing thickness, so that a gradient is created in the interior of the compartment, so that the Electrolyte solution - if necessary together with the separated protein - can be completely emptied via an outlet opening at the lowest point.

In the event that the compartment and the cooling element formed from capillaries form a structural unit (Fig. 2), each side part contains one or more recesses (openings). Due to the modular structure of a multitude of compartments, these cutouts form cooling water channels through which the cooling liquid flows in on one side - through the capillaries - and out of the opposite cooling water channel. It goes without saying that the electrophoresis chamber according to the invention - composed of individual compartments - contains connections so that the cooling water channels can be supplied with cooling liquid.

If it is desired or necessary to increase the cooling capacity, the capillaries of each compartment can be supplied with cooling liquid alone or combined in blocks, separately. The temperature of the cooling liquid can - for example using cryostats - be adapted to the separation problem that arises, for example in a temperature range between 1 to 30 ° C or in low-temperature electrophoresis between -10 to -30 ° C. When separating in the presence of high polyol concentrates, the preferred temperature range is around 20 ° C.

The capillaries are connected to the side parts in such a way that there is contact with the cooling water channel, but no cooling liquid can penetrate into the interior of the electrophoresis chamber.

REPLACEMENT The interfaces between two neighboring compartments are sealed against each other by a separating element. Accordingly, the edges of the side parts and the edges of the bottom part of each compartment are designed in such a way that they fulfill a sealing function and no electrolyte liquid can escape. For example, they can contain sealing profiles made of rubber or another suitable material - such as Teflon or silicone.

The separating elements serve to prevent liquid exchange between adjacent compartments, while the proteins to be separated can diffuse, i.e. the separating elements fulfill the function of a membrane. Substances that have this membrane function and are also sufficiently mechanically stable can be used. Suitable materials are, for example, porous polymer films, ceramic membranes, or technical fabrics that are coated with a very thin gel. Such fabrics are described for example in German Offenlegungsschrift 37 36 087, to which reference is hereby made.

Ultrathin tissue-supported polyacrylamide gels or agarose gels with a thickness of approximately 50 μm to 2 mm, preferably 50 to 100 μm, are particularly preferred. Such thin tissue-supported gels can be placed directly between two compartments without the need for further design measures to accommodate a separating element. As a result of an external

REPLACEMENT LEAF Pressure applied pressure fix the gels between the compartments. The area of the gels is dimensioned so that the cooling water channels are not covered.

In one embodiment according to the invention, it is provided that thin frames with a firmly connected fabric, on which the gel can be polymerized, are used as separating elements. This embodiment enables a quick construction of the

Electrophoresis chamber, or an easier exchange of the separating elements. It goes without saying that in this case the frames must have recesses for the cooling water cannula of the same size as the associated compartments.

It is possible that the gels contain additives as can usually be used in electrophoresis. This makes it possible to adapt the electrophoresis chamber according to the invention to the various separation problems posed. For example, when using

Polyacrylamide gels additional functional groups are introduced into the gel, as is also known in the art of isoelectric focusing in immobilized pH gradients.

The dimensionless capillary cooling system according to the invention can be in various embodiments. The preferred embodiment, in which the capillaries are firmly connected to the side parts of the compartments and are supplied with cooling liquid through cooling water channels, has already been described above.

REPLACEMENT In another embodiment, the capillaries of each compartment are connected to form an “endless” capillary and connected to a coolant system.

In a further embodiment, the capillary cooling system is not firmly connected to the compartment. The capillaries are arranged in a flat frame, which also contains the device for supplying and disposing of coolant. The frame is dimensioned so that it can be inserted into the grooves provided in the compartment. It is necessary that the frame with the capillaries is arranged parallel to the interfaces of the compartments.

The electrodes (cathode or anode) of the electrophoresis chamber are arranged in the first and last compartments. Although the design of the electrodes can be varied within the scope of customary embodiments, it has proven to be advantageous to use an electrode constructed in the manner of a network. A "coarse-mesh" mesh (1-3 mm mesh size) made of an inert material (eg plastic) is meandering through a conductive material, preferably a platinum wire. Nets that are braided exclusively from a platinum or platinum-iridium wire are also suitable. Graphite electrodes or titanium platinum electrodes are also suitable. The area of the electrode roughly corresponds to the inner area of the compartments. Because of the high field strength between 50 ml to 200 volts / cm and the associated gas development in the electrode space, it is advantageous if the two compartments that contain the two electrodes have a significantly larger volume than the other compartments. This avoids excessive foaming. The segmented structure of the electrophoresis chamber according to the invention enables the electrolyte solution of the compartments with the electrodes to have a different composition than the other compartments. For example, a polyol reduce foaming in the region of the electrode ^"a higher content.

The electrophoresis chamber according to the invention is composed of a large number of parts, with compartments and separating elements alternating. The two outer compartments contain the two electrodes. This layered electrophoresis chamber is held together by a tensioning device in order to seal the individual components (compartments and separating elements) in such a way that no electrolyte solution escapes to the outside and no liquid exchange can take place between adjacent compartments.

In the electrophoresis chamber according to the invention, it has proven advantageous for the anticonvective stabilization of the separated substances, the chamber with a 40-80% solution of a polyol, e.g. Fill glycerin, sucrose, sorbitol or a mixture of these polyols. The individual compartments are separated from each other using tissue-based polyacrylamide gels. With this anticonvective stabilization, it is possible to take samples from all segments during the separation and to elute the proteins easily after the separation without interfering mixing. Already with the introduction of isoelectric focusing in pH gradients stabilized by polyol density gradients, it was shown that high polyol concentrations are compatible with electrophoresis.

HE In particular with a high substance loading in the chamber, it may be necessary to constantly circulate the contents of the individual compartments with the aid of a multi-channel pump in order to prevent electrode decantation. Electrode decantation would cause separated substances to collect in the lower part of the compartments, which would impair the separation and is undesirable. For optimal separation, a uniform distribution of the separated substances and electrolytes over the entire cross-section of the compartments is desirable. The pump is excluded via the discharge channel of the individual compartments and the pumped liquid is returned to the upper part of the respective compartments via a hose.

Compartmenting with tissue-based polyacrylamide gels offers a number of advantages. Polyacrylamide gels are a matrix that is well known from analytical tests and does not interfere with isoelectric focusing and other electrophoretic separations. The layer thickness of the fabric-supported polyacrylamide gels can be chosen between 0.05-2 mm. In this way it is possible to variably adjust the ratio of the liquid phase to the gel phase in the separation chamber, which can have a decisive influence on the dissolution. The tissue-supported gels are mechanically stable and enable good compartmentation. The tissue-based gels can be washed, dried and rehydrated. The composition of the tissue-supported gels can be chosen as desired within certain degrees of crosslinking. The use of polyacrylamide gels also enables additional functional groups to be introduced into the gel, as is known from the technique of isoelectric focusing in immobilized pH gradients (Görg, A., Fawcett, JS and Chrambach, A, Adv. Electrophoresis 1988, 2 , 1-43).

The compartments can contain sensors for measuring important parameters, e.g. pH value, temperature, UV, IR, activity measurement of radioactive labeled samples, conductivity etc. The electrophoresis chamber according to the invention can be fully automated for discontinuous operation if an automatic sample application and sampling is additionally installed.

The electrophoresis chamber according to the invention is usually operated in a horizontal position. However, if 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.

To separate a protein, for example, the procedure is as follows:

One or more compartments of the compartments filled with electrolyte solution are emptied and filled with a mixture of the sample to be separated and the electrolyte solution. The course of the separation can either be followed by taking direct samples from the individual compartments or, if the compartments contain suitable sensors, by means of the measurement data obtained. The separated samples are isolated by simply emptying the relevant compartments.

REPLACEMENT LEAF 12

With isoelectric focusing, all compartments can be emptied and then filled with sample solution. The sample is then separated in the electric field according to the isoelectric points of the individual components.

The fractionation of carrier apholytes is an important sub-step in the production of carrier ampholytes for isoelectric focusing. With a high-resolution separation chamber, it should be possible to produce narrow pH ranges of carrier ampholytes with better defined properties than was possible with the previously usual methods. Such narrow pH ranges of carrier ampholytes are important for separations in which high resolution is required, such as is the case when examining genetic markers.

In several experiments, synthesis approaches of the carrier ampholytes were fractionated and the isolated fractions were examined in analytical focusing experiments. Thanks to the excellent cooling performance of the new separation chamber, the concentrated synthesis batch (35%) could be fractionated directly, ie without dilution, which could save the previously expensive concentration of the diluted fractionated carrier ampholytes. After a separation time of 44 h, the pH gradient shows an almost linear course, with a minimum conductivity characteristic for this pH range at neutral pH. In analytical refocusing, the pH gradients measured in the gel coincide with the pH range isolated in the preparative experiment, with sometimes excellent linearity over a narrow pH range (e.g. the range pH 3-4). The fractionation of carrier ampholytes shows that it is possible in the new separation chamber to carry out separations even with a high conductivity of the sample or the buffer electrolytes. Such separations could also be of interest for other low molecular weight substances.

To separate different proteins, the pH ^' gradient and the Vh product have to be optimized. This requires the pH gradients to be stable in the electrical field. In a test in pH 4-9 Servalyt carrier ampholytes, the pH gradient was constant for Vh products from 6000 - 13000 Vh. With preparative refocusing of narrow pH ranges, the pH range coincides with the originally isolated range. This experiment shows that cascade focusing is possible in two or more steps, which could significantly improve the resolution for components that are difficult to separate.

As shown in Figure 1, the electrophoresis chamber according to the invention is characterized by a modular structure. On a base plate (1), the compartments (2) are arranged one after the other, the separating elements which are located between two Ko artimenten not shown in this drawing. Following the compartments containing the electrodes (3) there are two stable end blocks (4) which have devices (5) for receiving two guide rails (6). The plates, the guide rails and the end part (8) allow the individual compartments to be fixed, so that a liquid-tight electrophoresis chamber is formed. The guide rails can, if they are designed as a round rod or as a tube, contain a thread.

REPLACEMENTB The end piece (8) is put on and screwed with the nuts, whereby the required pressure is exerted on the electrophoresis chamber via the end piece which can move on the guide rails. The end blocks (4) also serve to seal the recesses (7) for the two cooling water channels so that the cooling liquid does not leak. The end blocks also contain devices (9) for connecting the capillary cooling system (11) to a cryostat or another coolant supply (not shown in the drawing). Seals (12) between the compartments (2) prevent liquid from escaping. The recesses (7) form two opposite coolant channels. The electrical leads to the electrodes are also not shown in the drawing. The cohesion of the compartments can of course be achieved by means of appropriate technically equivalent devices.

With one exception, the compartments are only shown in elevation in order to show the structure of the electrophoresis chambers according to the invention more clearly.

In general, the chamber consists of at least 5 compartments, as a smaller number is possible, but only makes sense in exceptional cases from the point of view of the separation performance. Shortened separation sections with 5 or fewer compartments are useful if they are used in so-called cascades. The sample is first separated in a first electrophoresis chamber, the content of a compartment subsequently being fractionated further in a further electrophoresis chamber. This process can be repeated several times. The combination of several electrophoresis chambers with a small number of compartments in a cascade enables, for example, the rapid separation of a protein mixture.

Figure 2 shows a section through a compartment (2) with a permanently installed capillary cooling system. Two side parts (14) and a bottom part (15) are connected to form a frame (16). The bottom part has a different thickness, at the lowest point there is a closable drainage channel (17), via which a multi-channel pump can also be connected in order to circulate the contents of the individual compartments to avoid electrode decantation. Openings (7) are provided in the side parts. The cooling water channels are thus formed by combining several frame parts. The capillaries (18) are firmly connected to the side parts (14).

Figure 3 shows a section through a compartment (2) with subdivided openings (7a) and (7b) to form separate coolant channels. For more effective cooling, they can be charged with cooling liquid in opposite directions, for example. Figure 4 shows a separating element (19) with a permanently installed tissue-supported gel (20). The side parts (21) contain cutouts (22) for the formation of cooling water channels. The side parts (21), the bottom part (25) and an upper part (23) form a fixed frame (24) to support the tissue on which the gel is polymerized.

Claims

1) Device for preparative electrophoresis, characterized by a modular structure of the electrolyte chamber from a large number of compartments, the individual compartments being separated from one another by elements (separating elements) with membrane-like properties and each compartment containing a cooling element consisting of capillaries arranged in parallel.

2) Device according to claim 1, characterized in that the separating element consists of a polymer film with membrane-like properties.

3) Device according to claim 1, characterized in that the separating element consists of a tissue-based polyacrylamide or agarose gel.

4) Dimension-independent cooling system for preparative electrophoresis, characterized in that it contains cooling capillaries arranged in parallel and having a small outside diameter arranged at a short distance from one another.

5) Dimension-independent cooling system according to claim 4, characterized in that the capillaries consist of steel and are coated with an electrically non-conductive material.

6) Dimension-independent cooling system according to claim 4, characterized in that the capillaries consist of plastic. 7) electrophoresis chamber, characterized in that it consists of

a) at least 5 compartments provided with cooling capillaries arranged at a short distance apart,

b) two compartments for receiving the electrodes,

c) a device for supplying the capillaries with cooling liquid, and "

d) a device for holding the compartments together

consists.

8) electrophoresis chamber according to one of claims 1, 2, 3 or 7, characterized in that it contains devices ^" for mixing the electrolyte in the individual compartments to avoid the electrode decantation.

9) electrophoresis chamber according to claim 8, characterized in that it comprises a multi-channel pump for mixing the electrolyte in the individual compartments to avoid the

Contains electrode decantation in the individual compartments.

REPLACEMENT