JPS6177751A - Continuous electrophoresis apparatus - Google Patents

Abstract

PURPOSE:To increase the amount of separating processing of a charged material, by arranging one electrode at the central part of a container, arranging other electrodes radially with said electrode as the center, and separating an annular part between the electrodes by using films passing ions or electricity. CONSTITUTION:A top surface 2 and a bottom surface 3 of a cylindrical container 1 are closed. The inside of the container is concentrically partitioned by films 4 and 5. An electrode chamber 6, an electrophoresis chamber 7 and an electrode chamber 8 are provided in the container 1 from the center to the outside. The films 4 and 5 are made of a material, which passes small particles such as ions in a buffer liquid or electricity but does not transmit particles such as protein having large molecular weight. A positive electrode 22 and negative electrodes 23 are provided in the electrode chambers 6 and 8. The electrode 22 is arranged at the central part of the container 1. The electrodes 23 are concentrically arranged with the electrode 22 as the center. A charged material is supplied from a nozzle 15. A separated material is taken out of nozzles 13 and 14. Thus the processing amount can be improved.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a continuous electrophoresis apparatus IT for separating charged substances such as proteins, nucleic acids, cells, etc. using electrophoresis.

[Background of the invention]

Conventional charged substances such as proteins, nucleic acids, and cells (hereinafter referred to as
Separation and purification methods for proteins (described as a representative example) include electrophoresis, membrane separation, and liquid chromatography. The membrane separation method is a method for separating proteins based on the pore size t of the membrane, and allows for continuous processing, but has the drawback of poor protein separation ability. Liquid chromatography is a method of separating proteins by passing them through a carrier-packed column, and although it has excellent separation performance, it is a batch operation that does not lend itself to large-scale industrial-scale processing. Electrophoresis method.

This is a method of separating and purifying proteins in an electric field using the difference in charge of proteins. This electrophoresis includes a carrier electrophoresis method that uses a carrier such as a gel, and a carrier-free electrophoresis method that is performed in a free solution without using a carrier. Since the carrier electrophoresis method is a batch method, the carrier-free electrophoresis method is suitable for industrialization where large-scale processing is carried out because it allows continuous processing.

Support-free electrophoresis method 1 For details, see Kurt Hanni from West Germany.
ng literature (Wlectrophoresis.

P235-P243, 1982). The method is shown below.

The protein mixture to be separated is continuously injected from an injection port into a separation buffer solution flowing at a constant rate across an electric field in the electrophoresis chamber. Each protein has a different loading amount, so its movement speed in the electric field is different. Therefore, while flowing through the separation buffer solution, the particles are deflected and separated depending on the flow rate of the separation buffer solution. As described above, since this method can perform continuous separation, it is found to be effective for industrial-scale separation and purification of proteins. In order to improve the separation performance in this method, it is important to always keep the flow rate of the separation buffer solution in the separation chamber constant. However, Joule heat is inevitably generated when a current is passed through the separation buffer solution, and this heat causes a convection phenomenon in the separation buffer solution, which disrupts the flow of the separation buffer solution.

Therefore, the ability to separate proteins decreases. Therefore, in order to improve this point, conventional carrier-free electrophoresis devices require extremely precise control of the temperature and flow rate of the separation buffer to ±0.2%, and for this purpose, the separation chamber has to be made as thin and flat as possible. However, the throughput is small, which is a problem when scaling up to an industrial scale.

Sara again? In order to eliminate the effects of thermal convection, there is a plan to smuggle an electrophoresis device into space and perform electrophoresis without any force, which shows how big a problem this convection is.

[Purpose of the invention]

The purpose of the present invention is to provide a new continuous electrophoresis apparatus that can increase the throughput. Regarding the fact that the convection of the separation buffer liquid due to Joule %t disturbs the uniform flow n of the separation buffer liquid, this method is actively used to achieve the above purpose. It is. That is, according to a preferred embodiment 1 of the present invention, f1 to be separated in a waste container is
Continuous electrophoresis in which an electrically charged substance flows in, a voltage V applied between the electrodes causes the electrically charged substance to undergo electrophoresis, and thereby the separated substance is extracted! In Itit,
One electrode is placed in the center of the container, the other electrode is placed in a concentric circle around the one electrode, and the annular part between the electrodes is isolated using a membrane that conducts ions or electricity. and the annular portion P: at least one portion into which the charged substance flows.
2 nozzles are provided and the separated substances are taken out.
It is characterized by having more than one nozzle.

[Embodiments of the invention]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below using embodiments of the present invention shown in FIG.

First, an embodiment of the present invention will be described with reference to FIGS. 1 and 5i:l#. FIG. 1 shows a longitudinal sectional view of an embodiment of the invention. FIG. 1 also shows the flow of fluid inside the device. FIG. 2 shows the A-A arrow direction (cross-sectional view) in FIG. 1. In Figures 1 and 2, 1
is a cylindrical container with a closed top surface 2 and bottom surface 3 and a cylindrical body. This container l is preferably cylindrical, but it does not necessarily have to be cylindrical, and can be elliptical, polygonal, spherical, or the like. The interior of the container 1 is divided concentrically into three parts by membranes 4 and 5. That is, from the outer circumferential side of the container 1, the electrode chamber 8. Migration chamber (migration chamber) 7
．． and three parts of the electrode chamber 6 in the center. Membrane 4 and membrane 5 separate the ions in the fur solution (eg, Na, K, H).

Small particles such as OH) or substances that conduct electricity but do not allow large molecular weight particles such as proteins to pass through can be used. As such a film,
There are dialysis membranes, ion exchange membranes, cellophane, etc. for dialysis, but the type of membrane is not particularly important to those who have not yet invented it. Furthermore, the term "film" is used for convenience, and means not only a relatively thin film like a normal film, but also a considerably thick film.

9°10. 11, 12, 33, 14 and 15 all indicate nozzles. The nozzles 9 and 10 are for supplying and circulating the separation buffer solution to the electrode chamber 81. Nozzles 3 and 12 are for supplying and circulating the separated buffer solution to the electrode chamber 6. The nozzles 13 and 14 are for taking out the separated substance and constitute a take-out section. A plurality of nozzles serving as the extraction section are provided depending on the number of substances to be extracted. The nozzle 15 separates charged substances (proteins, etc.) into a container (strictly speaking, inside the migration chamber 7).
It is for supplying C, and constitutes a supply section. The pump 16 is for supplying and circulating the separation buffer solution into the electrode chamber 6. The temperature regulator 17 is for adjusting the temperature of the separation buffer solution supplied and circulated into the electrode chamber 6. The conduit 18 is a conduit for supplying and circulating the separation buffer solution. The membrane 19 is for supplying and circulating a separation buffer solution into the electrode chamber 8 . Temperature regulator 2
0 is provided to adjust the temperature of the separation buffer solution supplied and circulated within the electrode chamber 8.

The pipe line 21 is a pipe line for supplying and circulating the separation buffer solution. 22 and 23 are electrodes, which are installed in each electrode chamber 6.8. By applying a voltage between these electrodes,
A potential difference is created within the migration chamber 7, causing electrophoresis of the charged substance. Note that the electrode 22 is a positive electrode, and the electrode 23
is the negative electrode.

The electrode 22 is arranged at the center of the container 1, and the electrodes 23 are arranged in a plurality of concentric circles around the electrode 22. Basically, any electrode material may be used as long as it is a good electrical conductor, but it should be one that does not corrode or dissolve due to the separation buffer solution.

As such a material, platinum, platinum-plated titanium, etc. can be used. In FIG. 1, the broken line arrows in the electrophoresis chamber indicate the direction of flow of the separation buffer solution, and the solid line arrows indicate the direction of protein flow.

Next, using the apparatus shown in FIGS. 1 and 2, A, B2
Proteins A and B are extracted from a separation buffer solution in which different types of proteins are mixed (hereinafter referred to as mixed separation buffer solution).
Explain how to separate and extract. First, a mixed buffer solution is supplied from the nozzle 15 to the migration chamber 7. In addition, the pumps 16 and 19 are activated to supply the separation buffer solution to the electrode chambers 6 and 8t, and circulate it. Each portion of the E buffer solution is regulated with a temperature regulator 17.20. The temperature in this case is adjusted so that the temperature of the separation buffer solution in the electrode chamber 6 is higher than the temperature of the separation buffer solution in the electrode chamber 8. In this way, convection occurs more smoothly and stable separation processing can be achieved. A DC voltage is applied between electrodes 22 and 23.

As a result, the proteins in the mixed separation buffer solution injected from the nozzle 15 undergo electrophoresis within the migration chamber 7.

The charge state is different between protein containing and protein <iB.

Now, suppose that the equipotential of the danpo (pawn) is PI, the isocoordination of protein B is PIB, and there is a relationship t such that PI > PI. t so that it is an intermediate value of
Use the one that has been adjusted to v4. Under such conditions, the injected protein (pawn) moves to +& (electrode 22 side) and protein B moves to one pole (electrode 23 side) by electrophoresis. Also, the joules generated at this time Due to the heat and the temperature difference of the separation buffer solution described above, convective movement of proteins occurs.

In other words, the current density (iA, B) causes movement due to electrophoresis and movement due to convection.The current density increases the closer you get to the central electrode 22t, and the RI% also decreases, so the temperature rise due to Joule heat. The temperature is higher on the side closer to the center. Therefore, convection is more likely to occur, promoting the smooth movement of protein. The temperature control described above is
This is an auxiliary means of movement. In this state, protein A moves toward the nozzle 14, and protein B
The nozzle moves 13 inches in the opposite direction. Therefore, a buffer solution containing a large amount of protein B can be extracted from the nozzle 13, and a buffer solution containing a large amount of protein B can be extracted from the nozzle 14. PI as protein B, = 6
．． 85 myoglobin, PI as protein pawn, = 1
A mixture of zotium 1 and zotium was mixed in a boric acid-sodium hydroxide separation buffer solution with a pH of 9.0, and electrophoretic separation was performed using the same apparatus as shown in Figure 1. It is not possible to take out lysotium from nozzle 14 and myoglobin from nozzle 13. And nozzle 13',
As a result of comparing the case where one 14° and 15 nozzle is used and the case where two nozzles each are used, the processing is approximately twice as much when two nozzles are used, and the separation of #jI group There was no significant difference in accuracy between the two.

As is clear from the structure of FIGS. 1 and 2, a plurality of nozzles 13, 14, which are the take-out portions, can be provided radially from the center of the container. Note that if the temperature of the separation buffer solution flowing into the electrode chamber 8 on the outer circumferential side is made higher than the temperature of the separation buffer solution flowing into the electrode chamber 6C, the direction of convection becomes opposite to that in the above case.

Even in such a case, separation is possible. Furthermore, the polarity of the voltage applied to the electrodes may be reversed.

Next, another embodiment of the present invention will be described. FIG. 3 shows a longitudinal sectional view of another embodiment of the invention.

@Figure 4 shows a cross-sectional view of Figure 3. In Figure 3, the pump,
Temperature meters, etc. are omitted. In this example,
There is one electrode 23 arranged radially, and the shape is cylindrical. If the electrode chamber 8 is made into a cylindrical shape, the electrode chamber 8 will be 2AI, so the separation buffer liquid 1 supplied from the nozzle 9 will not be sufficiently supplied to the inside of the electrode. 23-b is provided. The adoption of the cylindrical electrode 23 improves separation performance and reduces throughput.

Accuracy increases.

Further examples of ponds are shown in FIG. 5 and fJS6.

Figure 5 is a longitudinal cross-sectional view of a continuous electrophoresis apparatus showing another embodiment of the present invention, including a cylindrical body, 2 a top surface, 3 a bottom surface, and a membrane 4.
Membrane 5. Nozzle 9 to nozzle 15. The structure and function of the main parts such as the $ pole and e pole are the same as in Fig. 1. Although the pump, temperature regulator, pipe line, etc. are all omitted in FIG. 5, the same ones as in FIG. 1 are connected.

However, the electrophoresis apparatus shown in FIG. 5 differs from the case shown in FIG. 1 in that a cylindrical current plate 24 is provided. @Figure 6 is a view seen from the BB cross section in Figure 5. As shown in FIG. It is arranged concentrically with the membrane 5. FIG. 7 is a front view of the current plate 24.

As shown in Fig. 7, it has a cylindrical shape, and has a cutout 24-a at the lower end, and an opening 24-b at the other part, and the area of each opening is , large incision lower part 2
4-a Make it smaller than the opening area per one piece. When the above-mentioned rectifying plate 24 is provided, as shown by the broken line in Fig. 5, the effect of separating and rectifying the upward flow and downward flow of convection is to prevent flow turbulence and improve separation performance. be.

The current plate 24 is a plate with holes in it in FIG. 5, but if the plate is made of porous material such as cloth or filter,
4-a is not necessary and only the notch 24-b needs to be provided.

Furthermore, in the above embodiment, a temperature difference was created between the liquid circulating in the two electrode chambers 6 and 8, but other mechanisms for creating a temperature difference, such as heating the liquid with a heater without circulating the liquid, were added. You may do so.

In the above-mentioned example, 2m class protein pawns were used.

Although the method for separating and purifying B has been described, it is also possible to put a liquid containing one type of protein into the electrophoresis chamber 7, concentrate it, and take it out from the nozzle 13 or the through hole 15.

This device also has the advantage of being usable as a concentration device. Of course, three or more types of charged substances can be separated by providing a large number of nozzles for single separation and extraction.

〔Effect of the invention〕

According to the present invention, the throughput can be significantly improved compared to the electrophoresis apparatus according to the prior art 1, and the apparatus can be made larger.

[Brief explanation of the drawing]

FIG. 1 is a vertical cross-sectional view of one embodiment of the present invention, FIG. 2 is a cross-sectional view of FIG. 1, FIG. 3 is a vertical cross-section showing another embodiment of the present invention, and FIG. 5 shows an embodiment of the pond of the present invention, FIG. 6 is a horizontal 1-fr side view of FIG. 5, and FIG. 7 is a detailed view of a part of FIG. 5. It is. 1... Container, 2... Top surface, 3...
...bottom, 4,5...crotch, 6...electrode chamber, 7...migration chamber, 8...electrode chamber, 9
, 10, 11, 12, 13, 14. 15... nozzle, 16... pump, 17... temperature regulator, 18... pipe line, 19...Pump, 20...Temperature: A#i device, 21...
・Pipe line, 22...electrode, 23...electrode, 23-8, 23-b...liquid passage, 24...
・・・・・・Rectifier plate, 5th port 6th port off view

Claims (1)

[Claims] 1. In a continuous electrophoresis device in which a charged substance to be separated is supplied into a container, electrophoresis is caused in the charged substance due to a potential difference between the electrodes, and the separated substance is taken out by this, one electrode is is placed in the center of the container, and the other electrode is arranged radially around the one electrode, and the annular portions between these electrodes are isolated using membranes that conduct ions or electricity, and the annular portion is provided with the 1. A continuous electrophoresis device comprising a supply section for supplying a charged substance and a take-out section for taking out a separated substance.