IL96743A - Process and apparatus for treating electrophoresis carriers - Google Patents

Description

96743/2 nmanopiw •'Kim Ϊ>Ί s oi> ιρηη τ>ί>ηη Process and apparatus for treating electrophoresis carriers BENDER & CO. Ges mbH S011355.16 "Process and apparatus for treating electrophoresis carriers" The invention relates to a process and an apparatus for treating electrophoresis carriers. More particularly it relates to a process and apparatus in which the liquid used for treatment flows along the electrophoresis carrier which is to be treated.

The analysis, determination or characterisation of colloidal and macromolecular substances, e.g. proteins, is simply and expediently carried out with the aid of electrophoresis. This well-known method of measurement comprises the steps of electrophoretic separation and after-treatment, by means of which the fractions of the sample are fixed on the carrier and prepared for subsequent evaluation. If the fractions are to be analysed or measured by optical methods, the after-treatment serves to make the fractions visible on the electrophoresis carrier. For this purpose a number of chemical reactions (e.g. staining and decolorising) are required. These reactions or procedures are separated from one another by washes. A number of electrophoresis carriers have to be washed out before being used in electrophoresis (in order to remove unwanted ingredients (e.g. monomers remaining from the polymerization)) and possibly have to be charged with water, buffer solution or reagent (e.g. urea) .

Conventional electrophoresis carriers are gels (e.g. agarose gel, polyacrylamide gel) . These are used either with solid carriers or with no additional carriers. The gels may for example be polymerised on films or poured onto sheets (for horizontal electrophoresis) or poured between two sheets (for vertical electrophoresis) . It is also possible to use gels reinforced with netting. The electrophoresis carriers used or currently available are generally of the dimensions, e.g. (in cm) : 30 x 20, 16 x 18, 3 x 8, 10 x 15, 7 x 15, 5 x 1 or 5 x 2. These electrophoresis carriers are used in all sorts of thicknesses in conventional methods. Cylindrically shaped electrophoresis carriers are also still in use. The carriers mentioned can in most cases be used for both one-dimensional and two-dimensional electrophoresis.

US Patent No. 4391689 describes an apparatus having a large number of tanks provided for the individual steps of the method of measurement. In this apparatus the electrophoresis carrier is mechanically conveyed from one tank to another. US Patent No. 4222843 describes an apparatus in which there is a drum which carries the electrophoresis carrier and repeatedly dips it into a tank provided underneath. The liquids needed for the reactions and washes are placed one after another in the tank. O87/05111 describes an apparatus for the after-treatment of electrophoresis carriers which consists of a container having on its lid a rotatable retaining device in which the electrophoresis carrier is clamped. This retaining device moves the gel in the tank and hence in the liquid provided.

All these apparatus imitate the old method which is still most frequently used, in which the electrophoresis carrier is manually moved from one liquid container to another for the individual steps of the after-treatment and agitated therein either manually or on a vibrating device. In the apparatus mentioned the batch process is used. The liquids are supplied discontinuously or the electrophoresis carrier is introduced, for each step, into the particular liquid required.

We have now been able to provide a process and suitable apparatus by means of which the treatment and more particularly the after-treatment or development of the electrophoresis carriers can be carried out easily, in a labour-saving manner and, if desired, automatically.

The invention generally relates to a process for treating electrophoresis carriers in which a liquid used for treatment is caused to flow around and over the electrophoresis carrier in contact with it.

The invention further relates to an apparatus in which electrophoresis carriers may be treated comprising a container with, in spaced relationship, inlet and outlet means for a liquid used in the treatment, in which in use the liquid flows through at least part of the container between the inlet and outlet means, and the electrophoresis carrier which is to be treated is located during treatment in that part of the container through which the liquid flows.

As used herein, the expression "container" is intended to include conduit or other means in which the carriers may be situated whilst treatment liquid flows.

If the treatment or after-treatment of the electrophoresis carrier comprises a number of steps, the respective liquids may be passed successively through the container. The process and apparatus according to the invention may be used both for the treatment of electrophoresis carriers before electrophoresis (e.g. for the above mentioned washing out and charging) and for the after-treatment or development (e.g. staining) of electrophoresis carriers on which the fractions of the separated samples are located.

In the process according to the invention for treating electrophoresis carriers, the carrier stays in the same container during all the treatment steps. The particular liquid required is introduced into the container and allowed to flow away. If the treatment step is a longer-lasting one, the liquid is passed continuously through the container for a suitably long period. The liquid is therefore allowed to or caused to flow around the electrophoresis carrier. The chemical reaction or washing process is thus made more intensive, since the flow action means that the liquid in contact with the surface of the electrophoresis carrier is constantly renewed. There is no need for mechanical movement of the electrophoresis carrier. The liquid is either constantly renewed or circulated.

The process according to the invention and the apparatus suitable for performing it are suitable for all the above mentioned electrophoresis carriers. It is advisable to keep smaller pieces in frames either individually or in groups.

The apparatus according to the invention for treating electrophoresis carriers or for the after-treatment of electrophoresis carriers on which the fractions of the samples being analysed are located has a container with inlet and outlet means for the liquids used for the treatment. The inlet and outlet means are located in various areas of the container, for example they may be provided at opposite ends to each other. The liquids flow freely through the volume of the container between the inlet and outlet means. During the treatment the electrophoresis carrier is situated in this part of the container through which the liquid flows. By regulating the pressure at which the liquid is introduced into the container it is possible to regulate the intensity of flow of the liquid. The apparatus is preferably constructed so that the direction of flow obtained is either substantially vertical or horizontal. By suitable arrangement of the inlet and outlet means in the container it is possible to achieve a direction of flow which runs between the vertical and horizontal. In a preferred arrangement a substantially vertical direction of flow is generated, the flow being directed upwards. If, for example, an SDS gel (polyacrylamide gel with added SDS [sodium dodecyl sulphate]) with no fixed carrier is introduced into an apparatus of this kind, the gel may be kept suspended in the liquid by the flow of liquid. If the flow rate is great enough, the electrophoresis carrier is aligned in such a way that the large areas of its surface are substantially parallel to the direction of flow of the liquid.

The electrophoresis carriers may also be retained in a specific position in the container by conventional retaining means. These retaining means grip the electrophoresis carrier at the top and/or bottom and/or sides. The retaining means may also be in the form of a frame. Such a frame may be constructed, for example, so as to contain one chamber or a plurality of chambers the walls of which are formed by a small-meshed grid. If the electrophoresis carriers are retained by such means or frames, these retaining means/frames are preferably held parallel to the direction of flow in the apparatus. If retaining means/frames are used it is also possible for treatment liquid to flow vertically from top to bottom.

The process, apparatus and function of the apparatus will now be more particularly described by means of the Examples and accompanying drawings which follow, which are by way of Example only.

Fig. 1 shows a simple embodiment of an apparatus according to the invention for treating an electrophoresis carrier. This apparatus consists of a cylindrical container (1) provided with an inlet tube (2) and an outlet tube (4) . The inlet tube (2) opens into the annular duct (3) . A grid (5) may be suspended in the container (1) . The grid (5) covers the entire opening of the container (1) . The inlet tube and outlet tube are provided with the necessary regulating element (not shown) . The outlet tube (4) is provided, at its end projecting into the container, with a sieve. The annular duct consists of a pipe running concentrically with the inner wall of the container. The upper half of the wall of the pipe is provided with openings (6) . The liquid for each treatment stage is supplied under pressure through the inlet tube (2) , passes through the openings in the annular duct into the container and flows out through the outlet tube (4) . Since the liquid passes under pressure through the openings in the annular duct into the container, a flow is formed. The pressure of the liquid is adjusted so that the electrophoresis carrier placed therein is kept suspended in the liquid without reaching the grid (5) . The grid (5) serves only to hold the electrophoresis carrier back and prevent it reaching the outlet tube should the flow become too strong as a result of an operational error.

Fig. 2 shows a cross section through the annular duct (3) . An opening (6) is a hole drilled through the wall of the tube extending radially at an angle of 45° to the base of the container.

Fig. 3 shows a plan view of the inlet tube (2) and the annular duct (3) . The openings (6) are arranged in a circle at the same height above the base of the container. The circle is located on that side of the tube of the annular duct which faces the centre of the container.

The size of the container is selected so that the electrophoresis carrier has just enough room (without being bent or kinked) in the part of the container through which the liquid flows. If the apparatus according to Figs. 1 to 3 is to be used, for example, for treating a piece of SDS electrophoresis gel measuring 7 x 15cm or up to 10 x 15cm, it is advisable to use a container with approximately the following dimensions: Internal diameter of container (1) : 180 mm Internal height of the container (1) : 240 mm Spacing of the outlet tube (4) above the base of the container: 180 mm Spacing of the annular duct (3) above the base of the container: 2 mm External diameter of the annular duct (3) : 120 mm External diameter of the tube of the annular duct (3) : 10 mm Internal width of the openings in the annular duct (3): 1 mm Number of openings in the annular duct: 10 The liquid is introduced under a pressure of at least 60 millibars. The electrophoresis gel is kept afloat in the liquid without being washed up against the grid (5) .

Fig. 4 shows an alternative embodiment of the apparatus according to the invention. This comprises a cylindrical container (7), which has a conical, downwardly extending base with an opening (10) and tube connection for the inlet and outlet means. An inlet tube (8) with an annular duct and an outlet tube (9) correspond to the analogous components in the apparatus according to Fig. 1. The cylindrical part of the container is separated from the conical part of the container by a protective grid (11) . This protective grid prevents the electrophoresis gel introduced into the container from obstructing the opening (10) . The inlet and outlet tubes are fitted with the necessary regulating means (not shown) .

In this alternative embodiment of the apparatus, the liquid can be drained rapidly and completely away from the container when desired through the opening (10) . For a short treatment step or for washing the container, the liquid in question can be drained away through the opening (10) and possibly fed in again.

Example l By way of an example of the use of the apparatus according to the invention, the Coomassie-blue-staining of an SDS electrophoresis gel (polyacrylamide gel with SDS (sodium dodecyl sulphate) added thereto) will now be described, using the embodiment of the apparatus shown in Fig. 4: After the end of electrophoretic separation the gel is placed without a carrier plate in the container (7) . The staining solution is supplied under pressure through the inlet tube (8) , drained away through the outlet tube (9) and circulated by pumping round in this way for 20 to 30 minutes. The flow of liquid thus produced keeps the gel floating in the liquid. The container is then totally emptied through the opening (10) . Then the decolorizing solution is introduced into the container through the inlet tube (8) for 30 minutes under pressure and drained away through the outlet tube (9) . Fresh decolorizing solution is supplied constantly. (It is also possible to recycle the decolorizing solution by including a regeneration stage.) The flow of the liquid causes the gel to remain afloat in the liquid. This process (decolorizing) is continued until the substrate is (approximately) colourless. Then the decolorizing solution is totally drained away through the opening (10) . Then a 2% aqueous glycerol solution is introduced through the inlet tube (8), drawn off through the outlet tube (9) and pumped round for 20 minutes in this way. Then the gel is taken from the container and dried.

Example 2 As a further example, the silver staining of an SDS electrophoresis gel (polyacrylamide gel with added SDS (sodium dodecyl sulphate) ) according to Oakley will now be described using the apparatus according to Fig. 4.

After the electrophoretic separation has ended, the gel without a carrier plate is placed in the container (7) .

Step 1 The decolorizing solution, used to remove the bromothymol blue (for characterising the front) is supplied under pressure through the inlet tube (8) , drawn off through the outlet tube (9) and recycled. The flow of liguid thus produced causes the gel to stay afloat in the liguid. This decolorization is carried out for 20 minutes. In order to remove all the decolorizing solution it is drained away through the opening (10) .

Step 2 The container (7) is then filled with deionized water and emptied again immediately. The water is supplied and removed through the opening (10) .

Step 3 10% glutardialdehyde solution is supplied under pressure through the inlet tube (8) and drawn off through the outlet tube (9) . The gel floats in the liquid. This treatment is carried out for 20 minutes, with the solution being recycled constantly by pumping. In order to drain it away completely the glutardialdehyde solution is drained away through the opening (10) .

Step 4 Washing with deionized water is carried out analogously to step 2. This washing is carried out at least 3 times.

Step 5 The gel is intensively rinsed with deionized water for 30 minutes by introducing the water under pressure through the inlet tube (8) and draining it away through the outlet tube (9) . Fresh deionized water is constantly supplied. Then the water is drained away completely through the opening (10) .

Step 6 Rinsing as in step 2.

Step 7 Ammoniacal silver nitrate solution is introduced into the container through the opening (10) until the gel is covered. The arrangement is left to stand like this for 15 minutes.

It is also possible to perform this step by supplying the silver nitrate solution under pressure through the inlet tube (8) and draining it away through the outlet tube (9) . The silver nitrate solution is recycled for 10 minutes.

After this treatment, in both alternative embodiments, the silver nitrate solution is drained away completely through the opening (10) . The container is briefly cleaned 3 to 5 times by supplying deionized water through the opening (10) and then letting it run away. If the tubes (8) and (9) have been used, these must also be thoroughly cleaned.

Step 8 The developer solution is supplied through the inlet tube (8) , drained away through the outlet tube (9) and recycled. After 5 to 10 minutes the developer solution is drained away through the opening (10) . (The retention time of the developer solution must be chosen to suit the particular sample.) Step 9 For half an hour, deionized water is supplied under pressure through the inlet tube (8) and drawn off through the outlet tube (9) . Fresh water is supplied constantly.

Other after-treatment steps may be added on in the same way. When the after-treatment has ended the gel is taken from the container and prepared for storage.

Example 3 Washing of a polyacrylamide gel which is, optionally, dried after the washing process has ended, thus becoming suitable for storage and rehydration: Step 1 After the polymerisation has ended, the polacrylamide gel (optionally a fabric-reinforced gel) is placed in the container filled with deionised water (according to Fig. 4) .

Then fresh deionised water is allowed to flow from the bottom to the top of the container for 30 minutes so that the gel remains suspended or afloat. In so doing, all the dissolved substances, preferably monomer acrylamide, monomer cross-linking agents and excess ammonium persulfate are washed away.

Step 2 After the washing has been carried out, the supply of demineralised water is stopped and the washings are removed through the opening in the bottom.

After the container has been completely emptied, it is filled with an aqueous glycerol solution and this solution is circulated by pumping round for 15 to 30 minutes. Then the solution is drawn off through the opening. Shortly before the solution is completely removed, the gel is taken from the container.

Step 3 The gel is placed onto a hydrophobic base, covered with a film of polyester and by slightly pressing it down uniformly the excess liquid is pressed out. Then, using conventional methods (drying by air or microwave) the gel is dried. Gels thus treated are suitable to rehydration, even after long storage.

Polyacrylamide gels set on a carrier may also be treated analogously.

The Examples may be carried out analogously using other electrophoresis carriers which may optionally be held in a frame, as described hereinbefore, or other retaining means.

The apparatus according to the invention may be modified in various respects.

The inlet means, in the form of an annular duct, as described in the above examples may also be designed in other forms known per se. An annular duct which is fixedly connected to the casing of the container may be provided. The annular duct with the outlet openings for the inflow of liquid may also be replaced by a sieve plate. In small containers, in particular, the annular duct may be replaced by a straight tube having the corresponding openings. Instead of such a tube, a slotlike opening may be provided in the wall of the container for the inflow and possibly for the outflow. This arrangement is also particularly advantageous if an electrophoresis carrier is treated in the horizontal position. The apparatus can then be constructed as follows, for example: the direction of flow of the liquid is horizontal, the container is of low height, the slots for the inlet and outlet of liquid are provided on opposite sides of the container, substantially on an extension of the longitudinal surface of the electrophoresis carrier.

The base of the container may be flat or domed.

If a plurality of electrophoresis carriers are to be treated simultaneously in one container, it is advisable to distribute the inlet and outlet of the liquid uniformly over that part of the container through which the liquid flows. This may be achieved for example by providing a plurality of annular ducts, inlet tubes or slots side by side (the number preferably corresponding to the number of electrophoresis carriers to be treated) .

The size of the apparatus is advantageously selected in accordance with the size of the electrophoresis carriers used. As a guide for the size of the container, the individual electrophoresis carrier should be surrounded on all sides by a layer of liquid at least 1 to 10 mm deep, preferably at least 3 to 5 mm deep.

If electrophoresis carriers of greatly differing sizes are to be treated in the same apparatus, it is advisable to construct the outlet ((4) in Fig. 1, (9) in Fig 4.) so that the height of the level of liquid can vary with the size of the electrophoresis carrier. This can be achieved, for example, by providing outflow openings at different heights in the container. For treatment of a small electrophoresis carrier, for example, one of the lower outlet openings is used.

Another possibility is to design the outflow opening to be movable over part of the height of the container wall .

For using the above mentioned frames, corresponding retaining devices are provided, e.g. rails into which the frames can be inserted.

The container of the apparatus may also be surrounded by heating coils/heat exchangers. The liquid may also be introduced into the container at the required temperature. Consequently, each treatment step can be carried out at the optimum constant temperature. Moreover, the results for series of tests can be standardised, thanks to the constant temperatures of the treatment. If the treatment is carried out at elevated temperature, the treatment time is reduced.

In order to aid the movement of the liquid, a stirrer (preferably a magnetic stirrer) can additionally be provided.

The treatment method described above may also be automated by having a suitable control device (e.g. a PC) to regulate the in-flow and out-flow of liquids.

The apparatus may also be constructed so that a plurality of containers as described above are provided, in which several treatments may be carried out separately from one another. Depending on the switching of the PC, these treatments may run parallel or totally independently. In numerous treatment methods for electrophoresis carriers, particularly the staining process, it is useful to provide storage containers for relatively concentrated reagent solutions in the apparatus. Before being introduced into the treatment container, these reagents are mixed directly in the inlet pipe or in a special mixing chamber in the inlet pipe with the corresponding quantity of solvent.

If a comparison is made between the conventional manual treatment or the known apparatus, and the process and apparatus according to the invention, the following advantages become clear: 1. Compared with the conventional treatment by carrying out the steps by hand, there enormous time saving for the analyst.

In the procedure according to the invention, the analyst has only to initiate the individual steps by switching the regulating means over, but in other respects the apparatus and treatment can be left to run. This regulating process may also be done by means of a computer. In the conventional manual process, frequent interventions are required. (The labour and time expended by the analyst are particularly great in the longer treatment steps, e.g. the rinsing operations between the staining and decolorizing steps, in which at least 5 rinses are needed, each taking 5-10 minutes.) In the process according to the invention, by contrast, rinsing is carried out continuously for 30 minutes without the need for the analyst to intervene.

The time taken for the individual treatment steps is also reduced, since the liquid in contact with the surface of the electrophoresis carrier is constantly renewed according to the invention by the flow action.

In the process according to the invention, initially a larger quantity of reagent is used, owing to the continuous method of operation. The consumption of reagent can be optimized by matching the size of the apparatus to the size of the gel which is to be treated. However, by regeneration of the used reagent (recycling) the consumption of reagent can be reduced to a level corresponding to that of conventional methods.

The apparatus according to the invention is simple in terms of construction and requires no mechanically movable parts. The apparatus is suitable for all electrophoresis carriers in common use. Even electrophoresis carriers of different sizes can be treated therein. It is also possible to treat several electrophoresis carriers at the same time, if they are secured in position (e.g. in a frame) as mentioned above. The electrophoresis carriers are simply placed in the container (or in the frame by means of which they are then placed in the container) . There is no need for any laborious mounting of the electrophoresis carriers, in for example movable retaining means.

Claims (1)

1. CLAIM : An apparatus for treating an electrophoresis carrier with at least one liquid, comprising a container having an inlet, though which liquid is introduced into the container, and an outlet, through which liquid is removed from the container, wherein liquid introduced into the container flows through that part of the container which is located between the inlet and the outlet, and wherein the electrophoresis carrier to be treated is located during the treatment in that part of the container through which the liquid flows so that it is contacted by said liquid, wherein the inlet is located in the lower part of the container and the outlet in the upper part of the container, so that liquid introduced under pressure produces a substantially vertical flow from bottom to top, keeping the electrophoresis carrier afloat in the liquid. For the Applicants DR. REINHOLD COHN AND PARTNERS