DE1517953A1 - Quantitative multiple gel electrophoresis apparatus - Google Patents

Description

Description. of the "Quantitative" for which I have applied for a patent Multiple Gel Electrophoresis Apparatus ".

The area of application of the apparatus is the separation of proteins and protein-like substances (proteins such as glycoproteins, lipoproteins, nucleoproteins, Hemoglobins, enzymes, protein hormones). A special area of application is in the Medicine the electrophoretic separation of proteins and proteins in body fluids, especially in the blood serum. The apparatus can be used in routine and research. at For the separation all media can be used in the apparatus that are in liquid Shape a. can be brought and then solidify to gel, e.g.

Agar gel, starch gel, in particular polyacrylamide gel.

The state of the art is as follows. In the case of "disc electrophoresis" from CANAL INDUSTRIAL CORPO @ PATION, 4935 Cordell Ave., Bethesda j4, MD (see Appendix No. 1) the tubes are filled with the medium in contrast to my apparatus individually. After separation, the gels are made by circumnavigating the gel cylinder separated from the wall of the tube with a hypodermic needle, leaving an intact gel surface is not guaranteed and errors occur in the densitometric evaluation. Evaluation of the stained gel cylinder is never done with a specially designed one only for this purpose constructed densitometer "Microdensitometer Model 8".

According to the company, densitometers from other companies are used for quantitative Evaluation of the gel cylinder cannot be used. I doubt gel cylinders such Thickness (5 n) can be precisely quantitatively evaluated are as any quantitative Evaluation requires the thinnest possible layer.

2. At the company SHANDON LABORTECHNIK GMBHt 6 frankfurt 1, Hafenstrasse 1, sold acrylamide column electrophoresis apparatus (see annex No. 2) is also a disc electrophoresis, the apparatus works according to the same principle as that specified under 1. Here are the same objections and to make comments, 3. From z-C APPARATUS CORPORATION, Philadelphia 4, Pa., 220 p. 4th St., an apparatus is used for the separation in polyacrylamide gels sold under the designation EC-470 (see Appendix 3). it serves according to information the company for analytical, preparative and vertical separation on large polyacrylamide plates. In contrast to the apparatus for which I have registered a patent, the production of the gel plates, which are particularly suitable for preparative separations, in a horizontal position Position made and only the electrophoresis itself carried out vertically, should several serum protein samples can be separated in such a gel plate and added to this Purpose to be applied at a certain distance from each other, so a "comb" in pressed the surface of the gel and placed a serum sample in each of the wells created in this way (8 in total). when applying the samples and by subsequent electrophoresis In contrast to my apparatus, these fractions are not through Partition walls separated from each other, resulting from the flour such between walls Fractions with little substance narrow and fractions with a lot of substance (e.g. albumin) wide zones across the direction of migration, but because the evaluation on the densitometer If the slit is of the same width, narrow and wide fractions may appear visually recorded differently, as a result of which also not evaluated quantitatively exactly. The partition walls in my apparatus result in different expansion which prevents factions, so they are all fixed in width. That guarantees a quantitative evaluation.

40 The company VITATRON N.V., Dieren / Holland, 23 Spoorstraat, offers an agar electrophoresis developed by Prof. Wieme (see Appendix 4), in which after Prof. Wieieme can also use polyacrylamide gel. The separation takes place here, in contrast to my apparatus, horizontally. Becomes a gel with a free surface manufactured so received but you don't get a uniform surface. Used if acrylamide is used, the surface exposed to the oxygen in the air does not polymerize well done. In addition, the polymerization is carried out in the absence of oxygen a nitrogen atmosphere too cumbersome, especially for routine examinations. The apparatus for which I have applied for a patent does not show these difficulties, since the vertical casting in narrow, square channels means that there are no large surfaces develop.

The "Quantitative Multiple Gel Electrophoresis Apparatus" continues composed of the following parts (Fig.A): I. The upper part (1,2,3), consisting of a hollow cylinder with a slot (1) to direct the electric field in a buffer container for approx. 1.5 1 ii; electrolyte fluid (buffer solution) with electrode (electrode hole (5)), the actual separation chamber, the parts of which (see below) by tightening the screws leading through the two-sided jaws of the buffer container be held together in such a way that no liquid escapes from the side.

II. The lower part (4), consisting of a buffer container with electrode, also for approx. 1.5 l of buffer solution, of such size that the lower piece the separation chamber is slightly immersed in the buffer solution.

The separation chamber consists of the following parts (7 @ lo): In the through the two outer, with the two upper buffer chamber halves connected, mirror image of the same bark with laterally adjacent cheeks limited to Available inner haunches are on both sides, from the outside to the inside, a PVC pane each (8) and plexiglass (10) milled out on one side. The two, with have plexiglass panes lying next to one another on their smooth sides on their machined, outer sides 20 identical, parallel, vertical millings with one Cross section of 1.5 x 7 mm as gel chambers (9). The four discs are made as a block in the inner space, in which a close-meshed plastic net (7) was previously placed is inserted from the top when the side screws are loosened.

The plastic net prevents the gels from sliding out of the gel chambers. The separation chamber is at the bottom for the filling process with a foam rubber and a plexiglass plate (3) of the same size as the bottom closed. Between the plastic net and the floor thus arises a cavity (6) as an extension of the perpendicular through the jaws leading filling channel (6), through which the simultaneous loading of all Gel chambers through the plastic net according to the communicating principle Tubing takes place. The bottom and separating chamber are secured by brackets attached to the side held together.

After the gel has formed, the bottom is removed and the upper part of the Apparatus for subsequent electrophoresis on the lower part of the apparatus set. The gel block created under the plastic net dips into the Buffer solution in the lower chamber, preparation of the apparatus for electrophoresis : After the separation chamber has been assembled, all el chambers are filled. Through the Filling channel so much sol is poured that all gel chambers up to about 1/2 cm below the top are filled. After the gel has formed, the soil is removed upper buffer container filled with buffer solution and the sample to be examined using Capillary pipette applied to the gel surface through the buffer solution. It should be noted that when the capillary pipette is blown out, mixing occurs of sample and buffer solution is avoided.

To increase the viscosity of the sample, approx. 10% glucose or urea can be used can be added. The amount of sample to be applied depends on the type of examination addicted. To separate the serum proteins, 0.03 to 0.004 ml of serum per gel strip are sufficient. At least one spirit tire is charged with bromophenol blue colored buffer solution, in order to determine the point at which the electrophoresis ended at its rate of migration To be able to determine Carrying out the electrophoresis: The electrophoresis is expedient either carried out in the cold room (+ 4 ° C) or cooling water or a Special cooling brine additionally in the two outer thick walls of the separation chamber attached channels (not shown). The electricity becomes a voltage. and current-constant power supply unit removed. The required amperage is e.g. for serum protein electrophoresis 65 mA at 120 to 150 volts. Electrophoresis takes a good hour under these conditions. Once the marker (Bromophenol blue) is approx. 1 cm from the lower end of the gel chambers, the Electrophoresis finished. Now the upper part of the apparatus with the separation chamber taken out of the lower buffer container and the upper buffer tank emptied. The separation chamber walls are removed by loosening the screws. After removal of the plastic net and the gel block underneath are the PVC panes carefully removed. The Gelitreifen are now exposed and are covered with a thin The spatula is pushed out of the millings into the staining solution, the staining solution consists e.g. in serum protein electrophoresis from a solution of amido black 103 in methanol-glacial acetic acid. The duration of the coloring depends on the dye used. For example, this takes Staining of the serum proteins with amido black takes only 1 to 1 1/2 hours. The jelly strips are then placed in the duck dye. After 2 to 3 times Renewing the decolorizer, the decolorization is finished after approx. 4 hours. The spirit hoops can now be quantitatively evaluated with a densitometer.

Presentation of the advantages that can be achieved with the apparatus over the State of the Teohnik: From the state of the art described at the beginning, the description the apparatus, the preparation of the apparatus for electrophoresis and the implementation electrophoresis, this apparatus has the following advantages: 1. 2 x 20 Gel strips can be cast vertically and simultaneously, and electrophoretically be separated. This eliminates what is sometimes tedious with other devices Fill each individual tube. In addition, such a large number was put into one sentence Proban to be treated not yet described.

2. There. The chamber is emptied in a single operation. Daa emptying single tube is cumbersome. No additional "gel column extractor" (see annex No. 5) is required. The difficult removal of the gel is no longer necessary. with a needle. Injuries to the gel are no longer possible.

3. The even. small layer thickness ensures a quantitative, densitometric evaluation. Caused by light scattering on curved gel surfaces i'errors in the evaluation are eliminated. lin further errors can occur in the evaluation of the ndisc electrophoresis "gels are caused by the fact that the gelsy after removal from the actual electrophoresis tube, staining and destaining Close a tube with a slightly larger lumen. For the purpose of evaluation, in which the gel cylinders are not well fixed and different when rotated Pherograms result.

4. through the partition walls are the individual gel strips of the same size separated from each other. The lateral limitation enforces the applied samples and separate fractions the same extent across the direction of migration. As well as Fractions containing larger as well as smaller amounts of substance have the same width, whereby a quantitative densitometric evaluation with the same optical slit is guaranteed.

50 The vertical, gel-filled millings provide the oxygen in the air With the exception of the small sample application area, no attack surface, so that too Polyacrylamide gels without air exclusion made from acrylamide by polymerisation can be.

6. The special construction of the apparatus enables 40 gels simultaneously produce 40 samples simultaneously reproducible the electrophoretic Subject to separation and quantitatively evaluate after staining and decolorizing.

Claims (3)

Claims. 10 gel electrophoresis * apparatus (Fig, A) for vertical separations, consisting of from an upper part (1,2,3) with buffer container, electron de and actual separation chamber, which is in a lower part (4), consisting of a buffer container with electrode, extends into it, characterized in that there is a special at the top of the apparatus Separation chamber is located through a fall channel on the principle of communicating The tubes can be filled in order to cell all forty gel chambers in one operation to enable. 2. Separation chamber according to claim 1, characterized in that it is used for Seal is held together by the side jaws of the buffer container. 3. Separation chamber according to claim 1, 2 and 3, characterized in that it is filled in a vertical position. 40 Trezmkammer according to claims 1, 2 and 3, characterized in that it is composed of several special plates (8,1o) that worked like this are that 2 x 20, separated by partitions, gel chambers (9) of rectangular Cross-section arise, the evenly thick, the applied samples and separated Fractions transverse to the direction of migration equally limiting, quantitatively evaluable Deliver gel strips.