CS658490A3 - Process and apparatus for treating electrophoresis carrier - Google Patents

Description

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The present invention relates to a process for the processing of a carrier for electrophoresis and to a device for carrying out the method in which the liquid used flows along the carrier.

The analytical determination of colloidal and macromolecular substances, for example proteins, is preferably carried out by electrophoresis. This known method of determination consists in electrophoretic separation and subsequent processing, the sample fractions being fixed on a carrier and thus ready for subsequent evaluation. In the event that the fractions are to be detected by optical procedures, the subsequent processing serves to make them visible on the support. To do this, chemical reactions such as coloring and discoloration are required. These reactions are separated from each other by the stages in which the carrier is washed. Some carriers must be washed before use, for example to remove unwanted components such as monomers remaining after polymerization, or because introduce water, buffer solution or a certain agent such as urea. Conventional electrophoretic carriers are gels, for example agarose or polyacrylamide. Tytogels are used as such or stored on a solid support. For example, gels can be polymerized into foils or a plate in the case of horizontal electrophoresis or poured between two plates in the case of vertical electrophoresis. It is possible to use gels reinforced by crosslinking. Suitable sizes of the carriers used are, for example, in cm: 30x20, lx18, 3x8, 10x15-7x15, 5x1 or 5x2. These carriers are used in a conventional manner at different thicknesses. It is also possible to use cylindrical carriers. In general, these carriers can be used for both single-direction and two-dimensional electrophoresis. The process according to the invention is suitable for all said carriers. In this case, it is expedient to place the smaller particles in the frames individually or in a larger number. U.S. Pat. No. 4,391,589 discloses an apparatus which contains a plurality of baths which are intended for individual assay stages. In this apparatus, the carrier advances mechanically from one container to the other. U.S. Pat. No. 4,222,643 discloses a drum-containing apparatus on which a support is mounted which is repeatedly immersed in the material in the container to be administered. Liquids that are required for processing are introduced into the container one after the other. In the patent application. WO 87/05111 discloses a post-processing apparatus comprising a container which is provided on its lid with a rotatable holding device in which the electrophoretic carriers are fixed. With this device, the gel moves into containers and liquids used. All these devices are influenced by their old-fashioned processes, in which the haymaker was manually transferred from one stage to another, from one container with a certain liquid to another container and rinsed manually or with the aid of a simple swinging device. Thus, it was a discontinuous process in which the liquids were fed one after the other, or the carrier was progressively transferred from one container to another.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method and an apparatus for processing and, in particular, subsequent processing of electrophoresis carriers in a simple, time-consuming manner and, if necessary, automatically. SUMMARY OF THE INVENTION The present invention provides a process for the treatment of electrophoresis carriers, wherein the liquid used for processing flows along the carrier. The invention also relates to an apparatus for carrying out the method comprising a container with an inlet and outlet for the fluid used, wherein the liquid flows in a portion of the container between the inlet and the outlet, and the support is in the flowed portion during processing. In the event that the processing or subsequent processing of the carrier is carried out in a plurality of stages, the corresponding liquids conduct the container in the appropriate order. The process of the invention and the apparatus may be used to process the carrier prior to electrophoresis, for example by washing or saturating with the above, or for post-processing, for example, dyeing carriers that already contain the sample sample. During the process according to the invention, the carrier is stored in the same container at all processing stages. The respective liquid is fed into the container and recirculated. If a stage is to last for a longer period of time, the liquid is allowed to continuously flow through the container during this smaller period. In this way it flows along the carrier. As a result, the chemical reactions or the washing process are intensified, since the liquid which is in contact with the surface carrier is still being replaced. It is not necessary to move the carrier mechanically. It is possible to feed the liquid constantly fresh or to let it flow in a closed circulation.

The device according to the invention for processing electrophoresis carriers of cells before or after the electrophoresis, wherein the carrier comprises a sample fraction is formed by a liquid inlet and outlet. The inlet and outlet are disposed at different locations of the container, e.g. The liquid flows freely between the inlet and the outlet. The carrier is in the region of the container through which the liquid flows during processing. The fluid pressure control can also control the fluid flow rate in the container. The device may be configured such that the liquid flows substantially vertically or substantially horizontally. By adjusting the inlet and outlet of the vessel, flow can also be provided in a vertical and horizontal direction. A device in which the liquid flows substantially vertically downwards is preferred. For example, if an SDS-gel (sodium dodecyl sulphate additive polyacrylamide gel) without a solid support is placed in such an apparatus, the gel is maintained in the fluidized bed by the flow of liquid. In addition, if the flow rate is sufficient, the carrier is disposed such that its large surfaces lie substantially parallel to the flow direction of the fluid.

The carriers for electrophoresis can be stored in a certain position with the aid of conventional holding devices. These aids are fastened to the carrier from above and / or underside and / or side. They can also have a frame shape. For example, the tytorams may be configured to form a chamber or a plurality of chambers, the walls of which are finely divided. When the support is held in these aids or frames, the devices or frames are preferably positioned parallel to the flow direction of the liquid. It is also possible to use these aids when flowing from top to bottom.

The invention will be further illustrated by the following examples with reference to the accompanying drawings.

Fig. 1 shows a simple embodiment of the device according to the invention.

FIG. 2 is a cross-sectional view of the annular inlet;

Fig. 3 shows a shoran view of the inlet and the annular inlet.

FIG. 4 shows a further embodiment of the device according to the invention.

Fig. 1 shows a simple embodiment of the device according to the invention for the processing of a single carrier. This device consists of a container 1 of a cylindrical body with an inlet 2 and a drain 4. The inlet 2 widens the annular inlet 3 A grid 5 may be suspended in the container 1, covering the entire upper opening of the container 1. The inlet 2 and outlet 4 are provided with the necessary controllers The outlet 4 is provided with a screen at its end extending into the container. The annular inlet 3 is formed by a tube which runs parallel to the inner wall of the container 1 and is provided with openings on the upper half. The liquid is fed through the inlet 2 at each stage of the process under pressure, exiting through the openings into the container 1, and then drained through the outlet 4. Because the liquid flows through the openings into the container 1 under pressure, flow is generated. The fluid pressure is adjusted so that the carrier floats on the liquid without reaching above the grid 5. The grille 5 merely serves to hold the carrier in the event that the flow is too strong due to the failure.

FIG. 2 shows a cross-section through the inlet 3; the opening 6 is a recess of the tube wall which extends radially at an angle of approximately 45 DEG with respect to the container 1.

Fig. 3 shows a view of the transverse 2 and the annular inlet 3. The openings 6 are provided at the same height above the bottom of the container 1. The ring is mounted on the side of the inflow tube 2 facing the central sleeve 1 "

The size of the container 1 is selected such that the carrier is accommodated in the flowing portion of the container 1 without bending. If the apparatus of Figures 1 to 3 is used, for example, to process an SDS-gel of 7 x 15 to 10 x 15 cm, the container 1 preferably has the following dimensions: inner diameter of the container 1 180 a and inner height of the container 1 240 aa flow-off distance 4 from the bottom of the container 180a and the distance of the annular inlet 3 from the bottom 2 and the annular inlet of the annular inlet 3 120a and the outside inlet aperture 2 10mm clear width of the openings of the annular inlet 3 4 1.

The liquid is fed under a pressure of at least 6 kPa '. This causes the gel to float on the liquid without rinsing the grid 5.

FIG. 4 shows a further embodiment of the device according to the invention. This device is formed by a cylindrical container 7 with a conical bottom provided with an opening 10 and an inlet tube 8 and a drain tube 9 of this part being analogous to the device of FIG. 1. The cylindrical part of the container 1 is divided by a protective grid 11 which separates the cylindrical part of the container 1 from its lower chamfer. This protective grid 11 ensures that the gel does not block the opening 10. Also, the supply tube 8 and the drain tube 9 are provided with corresponding control elements. In this embodiment, the liquid can be quickly and completely removed from the container 1 through the opening 10. In the case of a short treatment stage or in the case of washing out the container 1, the corresponding liquid can be introduced through the opening 10 and then removed if necessary. Example 1

The apparatus of Fig. 4 was used to stain Coomassie blue SDS-electrophoretic gel.

The gel was deposited without the carrier plate into the vial 7 after the electrophoresis. The dye solution was fed under pressure through the lance 8 and drained through the outlet pipe 9 and circulated through the pump for 20-30 minutes. During this time, the gel floated in the liquid. Then the vial 7 was completely emptied through the opening 10. Then the decolorizing solution was passed through the lance 8 under pressure for 30 minutes, which was drained through the drain pipe 9. Still fresh solution was fed in this case. it is possible to feed the solution in a closed circuit with the regeneration stage engaged. During this step the gel again 10 - in the liquid. This discoloration is carried out until the base layer (almost) is colorless. Then the solution is completely removed by the opening 10 and a 2% aqueous glycerol solution is fed through the inlet pipe 8, which is drained through the drain pipe 9a and allowed to flow for 20 minutes in a closed circuit. It is then gelled from container 7 and dried. Example 2 In the embodiment of Fig. 4, SDS-polyacrylamide gel is stained with Oakley silver.

After the electrophoresis is completed, gelled into the vial 7 without the support plate.

Stage 1

The decolourising solution used to remove the bromothymol blue (front marking) is fed under pressure through the inlet pipe 8 and discharged through the outlet pipe 9 in a closed circuit. During this procedure, the gel floats in the liquid. The procedure lasts 20 minutes, then the decolorizing solution is discharged through the opening 10. 11 -

Stage 2 The vial 7 is filled with deionized water and emptied immediately. Water is also fed through the opening 10.

Stage 3

A 10% glutardealdehyde solution is fed into the lance 8 under pressure and drained through the outlet pipe 9. The gel floats in the liquid. The process takes 20 minutes, the solution flows in a closed circuit using a pump. Then the seroztok discharges through hole 10.

Stage 4

The rinsing of the container 7 with deionized water is carried out in a similar manner to that in Step 2. The rinsing of the container 7 is carried out at least three times.

Stage 5

The gel was rinsed vigorously for 30 minutes with deionized water, the water was fed under pressure through the inlet tube 8 and drained through the drain pipe 9. The still-deionized water was fed, the water was then drained completely through the opening 10. 12 -

Grade 6

Rinsing the canister is performed as in step 2.

Grade 7

A solution of silver nitrate in ammonia 7 is fed into the container 7 through the opening 10. The solution is rendered so that the gel is covered. The solution is then allowed to stand in a 715 minute vial.

This step can also be carried out by bringing the silver nitrate solution under pressure through the feed tube 8 and withdrawing it through the drain tube 9. The tube then takes 10 minutes to circulate the solution. In any case, the silver nitrate solution is then completely discharged through the orifice 10. The container 7 is then rinsed briefly 3 to 5 times, with deionized water being supplied and discharged through the opening 10. If the lance 8 and the drain pipe 9 were used, these parts of the plant should also be thoroughly cleaned.

Stage 8 A delivery solution is fed through the inlet tube 8. The solution is drained through the drain pipe 9 and flowed in a closed circuit. After 5-10 minutes, the solution - 13 - is drained through the opening 10. The treatment time in this step is controlled by the sample to be treated.

Stage 9

The deionized water is fed continuously through the inlet pipe 8 under pressure and discharged through the drain pipe 9.

In an analogous manner, further processing steps can be included. After processing, the gel is removed and the evaluation is prepared. Example 3

The device can also be used to process a polyacrylamide gel prior to use for electro-foresis.

The procedures described in the example section can also be used to process other carriers, in the above-mentioned frames or by using holding aids.

The device according to the invention can be modified and supplemented in various ways. An annular feed, as described above, may also be provided in a different manner. It may be an annular inlet firmly connected to the container wall. The holes may also be replaced by a screen with a screen. Especially in smaller containers, the annular feed can be replaced by a straight-shaped tube with corresponding openings. Slots may be provided in place of the apertures for supply and, where appropriate, for the discharge of liquid in the vessel wall.

This arrangement is particularly suitable when the carrier is processed in a horizontal position. Thereafter, the device may be arranged such that the liquid flows horizontally, the container is low in height, the fluid inlet and outlet slots are disposed on opposite sides of the container in an extension of the longitudinal direction of the carrier.

The bottom of the container may be straight or wavy. If a plurality of carriers are to be processed in one container, it is expedient for the fluid supply and outlet to be arranged such that the liquid is evenly distributed in the container portion. This can be achieved, for example, by storing a plurality of annular inlets, inlet tubes or slots side by side, the number preferably being equal to the number of carriers.

The size of the device preferably corresponding to the type of carrier being processed. The carrier should be surrounded by all sides with a liquid layer of at least 1 to 10, preferably 3 to 5 on. If the carriers are of very different sizes and are to be processed in the same apparatus, it is expedient to provide a drain in the apparatus of Fig. 1 of Fig. 4 so that the height of the liquid layer can be adjusted depending on the size. carriers. This can be achieved, for example, by providing outlet openings at different heights of the container. The lower spout is then used to process the small carrier. Another possibility is that the outlet opening is slidably disposed in a portion of the height of the container wall. By using the aforementioned frames, various holding devices, for example rails, can be used, from which the frames are slidably supported.

The apparatus may also be provided with a heating coil or heat exchanger. In addition, a liquid of different temperatures can be introduced into the device. Thus, each stage can be carried out at an optimum constant temperature. The temperature average can also be adjusted with respect to serial investigations. When using a higher temperature, a shorter processing time can be achieved. For better fluid movement, a mixing device, preferably a magnetic stirrer, may be provided.

Postiip can also be automated so that the liquid inlet and outlet are controlled by a respective control unit, for example a computer. - 16 -

The apparatus may also be configured to contain a plurality of containers as described above, so that a plurality of treatments may be performed at the same time. According to a modification, for example by computer, these treatments can be carried out simultaneously or completely independently. In a number of processes for the treatment of carriers, especially in the case of dyeing, it is expedient to include a container for relatively concentrated reaction solutions. These reactants are mixed directly in the feed line or in a separate mixing chamber with the appropriate amount of solvent prior to feed into the container.

Compared with conventional manual processing or with known apparatuses for this treatment, the method and apparatus of the invention have the following obvious advantages. 1. Compared to manual processing, a lot of time is gained by a specialist. In carrying out the process according to the invention, the worker starts the steps only by switching the control unit and no longer has to interfere with the processing. On the other hand, the handling process requires frequent handling of the carrier, especially the longer-lasting stages, such as discoloration and flushing between the dyeing and bleaching stages, at which the rinse aid must be rinsed at least 5 to 10 minutes at least five times. In carrying out the process according to the invention - 17 - opposite the toau carrier, it is continuously rinsed without any need to be present. 2. The processing time in the individual stages is reduced due to the fact that the fluid is still renewed by the flow. In carrying out the process according to the invention, in continuous operation, the size of the apparatus can be optimized depending on the size of the processors, thus optimizing the consumption of reagents. procedures. The device according to the invention is simple and does not require the use of any mechanically movable parts. It is suitable for all common carriers and also for different sizes of these carriers. At the same time, it is possible to process a larger number of carriers if they are suitable for example in a frame. Placing the carriers in the device is simple, for example, when using frames, retractable shelves. For example, the mounting of the carriers in the movable holding aids is difficult.

Claims (6)

- 18 - ... G ..... PATENT CLAIMS A method for processing a carrier for IV electrophoresis, characterized in that the liquid "used for processing flows along the surface of the carrier" 2. The process according to claim 1, wherein the process is carried out in a plurality of stages, characterized in that the liquids used for processing are fed one after the other and flowed along the surface of the support. 3. Apparatus for carrying out the process according to claim 1, characterized in that it comprises a container (1, 7) with an inlet (1, 8) and an outlet (4, 9) for the liquid used, wherein the liquid flows through the container region (1, 7) between these parts and the carrier are deposited in this section of container (1, 7) during processing. 4. Apparatus according to claim 3 for the treatment of carriers in a plurality of stages, characterized in that it comprises a container (1, 7) with an inflow (2,8) and a drain (4, 9) for the liquids used, , 8) and the outlet (4,9) are provided in different portions of the container (1, 7), fluidly flowing one after the other through a portion of the container (1, 7) between its portions and the support is deposited in this container portion (1, during processing) 7). - 19 - 5. Apparatus according to claim 3 or 4, characterized in that the inlet (2, 8) and the outlet (4, 9) are arranged so that the liquids or liquids flow substantially or horizontally. 6. Apparatus according to claim 3 or 4, characterized in that the inlet (2,8) and the outlet (4, 9) are arranged in the lower and upper parts of the container (1, 7) so that the liquid fed under pressure flows from bottom to top. 60 7J99 / KO They represent FROM