DE3034899A1 - Continuous electrophoresis appts. has tubes for gel - fitted in with split sealing rings using handling device - Google Patents

Abstract

Appts. for continuous electrophoresis has containers, esp. glass or plastics tubes, filled with a gel, and sepg. the buffer spaces of the appts. Each tube is fixed in the appts. with a threaded ring possessing a slit; when the tube has been filled with the gel, a pipe having a helical thread at one end and an elastic ring is fitted over it. The fig. shows this pipe, acting as a holder, in the upper part and the tube contg. the gel in the lower part. Pref. the ring is of silicone rubber or perbunane. Pref. the pipe or holder is of plastics, esp. 'Plexiglass'.

Description

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Electrophoresis process, i.e. separation of different electrically charged Particles (ionized atoms and molecules) in the electric field, play in the analytical and preparative chemistry play an important role. Among numerous technical and principled In modern biochemistry, variants are most often used by ID i s k electrophoresis for purity testing and identification of biologically relevant molecules, in particular of proteins, used as a highly sensitive method. The procedure is with regard to pH value, buffer composition and gel size of the carrier are mostly used Polyacrylamide is based on a discontinuous separation system. Because of these discontinuities can be very narrow in practicals before the actual start of separation of the mixture and at the same time highly concentrated starting zones, which are responsible for the sharpness of the separation are crucial, generate. The high resolution of disk electrophoresis is caused by the initial concentration effect in the stacking gel and the subsequent Molecular sieve electrophoresis effect in the separation gel.

The latter causes a separation of the molecules not only according to theirs total electrical charge, but also according to its size (molecular weight) and Shape (conformation). Sign and magnitude of the electrical charges on protein molecules can be set by changing the solvent pH value (mostly buffer systems) will.

Figure 1 shows schematically the structure of a conventional disk electrophoresis apparatus. Two buffer reservoirs, upper and lower, stand over a row of vertically arranged ones Containers, mostly glass tubes, in connection with one another. The pipes (running pipes) included each collecting gel (upper third of the tube) and separating gel (lower two thirds of the tube). To start up the device, the lower reservoir is first filled with buffer solution filled. The samples to be examined are then applied to the stacking gel surfaces from above of the individual tubes.

The apparatus finally opens after carefully filling the top Reservoirs combined with buffer solution and placed under electrical voltage. Overall, the buffer systems are chosen so that the particles are present as anions and move towards the lower anode compartment The construction takes place at least for the moment from above to uiiten.

The devices previously used in laboratories and trade Disk electrophoresis have the following disadvantages: 1) Insertion and removal of the gel containers are complicated and hourly and require almost complete dismantling of the equipment, manipulations can only be carried out when the electrical fall is switched off: take place.

2) After switching off the current, the substance zones widen very quickly in the gel by diffusion: fractions that are close together rub into one another, and the sharp separating effect previously achieved becomes less frequent.

3) It is impossible to take samples with simultaneous trouble-free, continuous flow of the process. When dealing with unknown, identifiable Substances are but occasional sampling of great importance to the behavior the connection and quickly get a grip on the optimal test conditions.

All of these disadvantages are eliminated by our invention of specific devices eliminated.

The invention relates to a device for continuous electrophoresis, characterized in that the container filled with a gel, the two buffer spaces separates sealingly from one another by means of an elastic ring, the container in the device each with the help of a screw ring, which has a slot contains, are fixed and the container filled with gel at the top with a Tube, which at one end with a screw thread and an elastic ring is provided, are slipped over, The invention also relates to a method for continuous Ele: .- trophoresis, characterized in that the container filled with a gel with the help of a special device in the electrophoresis device, wherein the sealing of the container against the two buffer spaces by means of a elastic ring takes place, the fixation of the container in the electrophoresis device with the help of a the screw concentrically surrounding the container ensured then the upper buffer space by means of a screw thread at one end and a tube provided with a sealing ring opposite the gel-filled container seals and the gel-filled container under electrical voltage of the device removes.

PRINCIPLE OF THE DEVICES The disk electrophoresis apparatus is in Figure 2 shown. The lower and upper buffer reservoirs are over 12 glass tubes, which are filled with gel, connected to each other. Each tube is on top with fixed by a seal in the middle part of the apparatus. A drainage of the upper buffer solution along the outside of the glass tubes into the lower container is thus excluded. During the electrophoresis process, the middle part is filled with cooling brine or tap water, flushed through depending on the cooling requirement and thus the amount released during the electrophoresis process Joule heat dissipated. Platinum wires are used as electrodes, which are in the two Buffer spaces at the bottom and in a circular manner, to: central puit (apparatus growth) concentric are arranged.

Figure 3 shows a gel tube, from an elastic sealing ring and a retaining screw uneven. The pipe is pushed into a key, which is hollow inside and also contains an elastic ring. This ring is dimensioned so that it can hold the gel tube stably in the vertical position Is able to, but on the other hand releases it again if necessary when train. The lower Part of the key is made of V2A steel, the handle is made of PVC. When manipulating With the device, the two metal tongues grip into the slots of the screw ring as shown in Figures 4 and 5.

The introduction of a gel tube into the electrophoresis apparatus as well its fixation is shown in Figure 6. In Figure 7 is an already fixed Gel tube shown.

To remove a gel container, the top, i. E.

the side of the corresponding one protruding into the upper buffer tank The gel tube is sealed off from the upper buffer space (see AD formation 8). this takes place by means of a tube, which at one end has a screw thread and a Sealing ring contains (see Figure 9) t This is initially located in the interior Rohres buffer solution, which with the buffer solution outside before screwing the The pipe communicates The solution is extracted from the interior by means of a suction device (Pipette or similar) removed, and the gel tube can now with the already described The key can be removed from the appliance in the opposite way to when it was inserted. Instead of the gel tube, one uses, again with a sealing ring and Glass rod with a screw ring or a plastic rod. This is used for sealing the opening that has become free. This handling is also carried out with the aforementioned key. The sealing pipe can now be unscrewed and used to remove further gel pipes can be used in the same way.

Both the introduction and removal of the gel container can be dangerous with ongoing AD repair. be carried out under electrical SnannunC. Of the As a result, the electrosphoresis process will not remain sniffed, zc the substance zones by maintaining continuity, as shown in Figures 11 and 12 is.

Figures 11 and 12 show the different migration speeds a fraction of the enzyme ß-galactosidase contaminated with foreign proteins, in Dependence of different electrophoresis times.

The samples were prepared according to the method described in the apparatus 300 volts taken and evaluated. Note the sharpening of each one Substance bands.

LEGENDS TO THE DRAWINGS Fig. 1 Schematic representation of a usual disk electrophoresis apparatus Fig. 2a Side view of the disk electrophoresis apparatus, consisting of upper and lower buffer reservoir as well as one containing 12 gel tubes, coolable middle part Fig. 2b Disk - electrophoresis apparatus, top view, with arrangement of the 12 gel tubes. to the respective electrode in the center of the lower or upper Current feeds arranged in the external space are visible. Fig. 3 Msnipulation key (above) for inserting and removing the gel tubes. Gel tube (below) with seal and Fastening screw Fig. 4 Seal, fastening screw and lower part of the manipulation key Fig. 5 Manipulation key, holding the gel tube. The two tabs of the key are locked into the two slots of the fastening ring Fig. 6 Fastening procedure of a gel tube Fig. 7 Gel tube fastened in the apparatus Fig. 8 Gel pipe with a sealing pipe screwed over it for removal Fig. 9 Sealing pipe made of Plexiglas Fig. 10 Removal of a gel tube using a manipulation key and keys are hermetically sealed from the liquid through the sealing pipe the upper buffer space separated Fig. 11 Example of a disk electrophoresis run of the enzyme ß-galactosidase (molecular weight approx. 500,000 Dalton) under continuous The first withdrawal took place after 30 minutes and was in the period repeated between 30 and 90 minutes. I) The increasingly sharp separation of the fractions with increasing time is visible Fig. 12 Enlargement of Fig. 11 Blank page

Claims (6)

PATENT CLAIMS 1) device for continuous electrophoresis, characterized in that the container filled with a gel, the two buffer spaces separates sealingly from one another by means of an elastic ring, the container in the device each with the help of a screw ring, which has a slot contains, are fixed and the container filled with gel at the top with a Tube, which at one end with a screw thread and an elastic ring is provided, are slipped over. 2) Device according to claim 1, characterized in that the with Gel-filled containers made of glass or plastic are. 3) Device according to claim 1, characterized in that the elastic Ring made of Perbunan or Siliko £ {ouchuk is. 4) Device according to claim 1, characterized in that the tube is made of plastic. 5) Device according to one of claims 2 or 4, characterized gekennzeic net that the plastic is plexiglass. 6) method for continuous electrophoresis, thereby gekennzeic net that the container filled with a gel with the help of a special device introduces into the electrophoresis device, the seal of the container opposite the two buffer areas takes place by means of an elastic ring, the fixation of the container in the electrophoresis device with the aid of a Container Monzentrisch surrounding screw is guaranteed, then the upper one Buffer space by means of, at one end with a screw thread and a sealing ring provided tube with respect to the gel-filled container and the with Gel-filled container is removed from the device under electrical voltage 7) Procedure according to claim 6, characterized in that one is used as a special device internally provided with a hollow rod used, which in the hollow space an elastic Contains ring that holds the gel container in the vertical position and / or in the same Way releases and the slotted screw through two lateral Tongues moved.