Protein free flow isoelectric focusing electrophoretic separation device

Abstract

The utility model discloses a protein free flow isoelectric focusing electrophoretic separation device which comprises a cold liquid plate, a silica gel heat conduction pad is arranged at the top of the cold liquid plate, an electrophoretic separation chamber is arranged at the top of the silica gel heat conduction pad, the electrophoretic separation chamber comprises a separation chamber lower plate, the separation chamber lower plate is connected with the silica gel heat conduction pad, and a separation chamber upper plate is arranged above the separation chamber lower plate. The top of the separation chamber upper plate is connected with a pair of symmetrical electrode chambers, carbon rods are connected in the electrode chambers in a penetrating mode, and rectangular holes of the same size are formed in the bottoms of the electrode chambers and the top of the separation chamber upper plate. An anion exchange membrane and a cation exchange membrane which are used for isolating an electrode solution from an operation buffer solution are respectively adhered to the positions, corresponding to the rectangular holes, of the bottom of the separation chamber upper plate, a silica gel gasket is arranged between the separation chamber upper plate and the separation chamber lower plate, and the separation chamber upper plate and the separation chamber lower plate are connected through a clamp; a plurality of separation chamber liquid inlets and separation chamber liquid outlets are respectively formed in two sides of the short edge of the separation chamber upper plate.

Description

TECHNICAL FIELD

[0001] The utility model relates to protein separation technical field especially relates to a protein free flow isoelectric focusing electrophoresis separation device. BACKGROUND

[0002] With the continuous development of biotechnology and life science research, protein separation and purification technology has become an important issue in life science research. Isoelectric focusing (IEF) as a kind of efficient separation method, because it can be based on the isoelectric point (pI) of protein high-resolution separation, is widely used in proteomics, clinical diagnosis, drug development and other fields. Traditional isoelectric focusing technology generally adopts fixed medium (such as gel or membrane) to maintain the gradient of electric field, and makes protein focus according to its isoelectric point in pH gradient through electrophoresis. However, the use of fixed medium limits the flexibility and precision of separation process, and is low in efficiency in high-throughput analysis. In recent years, protein free flow isoelectric focusing technology (Free-Flow Isoelectric Focusing, FFIEF) as a new type of separation method arises at the historic moment. This method does not rely on solid medium, but through the electric field action in electrophoresis separation chamber, makes sample migrate and focus along the pH gradient in flowing liquid medium. Compared with traditional method, this technology has many advantages, such as higher resolution, less sample loss, better repeatability and greater sample processing capacity. Especially in the processing of complex samples and large-scale separation, protein free flow isoelectric focusing technology shows its unique advantages.

[0003] However, the existing protein free flow isoelectric focusing electrophoresis device generally has problems of inaccurate temperature control, low separation efficiency and complex equipment structure. These problems seriously affect the stability and repeatability of separation results, and in actual application, the operation is difficult, and problems such as uneven electric field and protein denaturation are easy to occur, which further reduces the separation effect. UTILITY MODEL CONTENTS

[0004] In view of the above problems, the utility model provides a protein free flow isoelectric focusing electrophoresis separation device, which effectively solves the problems of inaccurate temperature control, low separation efficiency and complex equipment structure of the existing protein free flow isoelectric focusing electrophoresis device.

[0005] The utility model discloses the following technical scheme: a protein free flow isoelectric focusing electrophoresis separation device, including cold liquid board, the cold liquid board top is provided with silica gel heat conduction pad, the silica gel heat conduction pad top is provided with electrophoresis separation chamber, the electrophoresis separation chamber includes separation chamber lower plate, the separation chamber lower plate is connected with the silica gel heat conduction pad, the separation chamber lower plate top is provided with separation chamber upper plate, the separation chamber upper plate top is connected with a pair of symmetry electrode chamber, the electrode chamber is connected with carbon rod and passes through, the electrode chamber bottom and the separation chamber upper plate top all are provided with the rectangular hole of same size, the separation chamber upper plate bottom is located rectangular hole correspondence respectively and is bonded with the anion exchange membrane and cation exchange membrane for insulating electrode liquid and running buffer solution, the separation chamber upper plate and the separation chamber lower plate are provided with silica gel gasket, the separation chamber upper plate and the separation chamber lower plate are connected through the clamp, the separation chamber upper plate short side both sides are provided with a plurality of separation chamber liquid inlet and separation chamber liquid outlet respectively.

[0006] Further, the cold liquid plate is circumscribed with a low-temperature cooling circulating pump.

[0007] Further, the electrode chamber top both sides are provided with a circular hole for electrode liquid circulation, the two circular holes are connected with an electrode liquid container and an electrode liquid driving pump through a pipeline, one end of the carbon rod above the anion exchange membrane is connected with a negative electrode of an external electrophoresis instrument power supply, and one end of the carbon rod above the cation exchange membrane is connected with a positive electrode of the external electrophoresis instrument power supply.

[0008] Further, the separation chamber liquid inlet is connected with a gas-liquid buffer chamber through a pipeline, the gas-liquid buffer chamber is filled with a background buffer solution, and the separation chamber liquid outlet is connected with a collection device through a pipeline.

[0009] Further, the electrode liquid container for cyclic exchange with the electrode chamber above the anion exchange membrane contains an alkaline solution, and the electrode liquid container for cyclic exchange with the electrode chamber above the cation exchange membrane contains an acidic solution.

[0010] Further, the thickness of the silica gel gasket, the anion exchange membrane, and the cation exchange membrane is 1 mm.

[0011] Further, the separation chamber lower plate is made of glass, and the separation chamber upper plate is made of acrylic.

[0012] Further, one of the separation chamber liquid inlets is connected with a protein sample to be separated through a sample driving pump.

[0013] The utility model discloses a protein separation device and method, which can precisely control the electric field distribution in the electrophoresis separation chamber, focus the protein according to its isoelectric point (pI), effectively control the gradient of the electric field through the circulation of acidic and alkaline solutions, maintain the low-temperature environment required in the electrophoresis separation process through the cold liquid plate and low-temperature cooling circulating pump (5), prevent the protein sample (11) from denaturation or degradation, ensure the stability and accuracy of the experimental results, isolate the electrode solution and buffer solution through the anion exchange membrane and cation exchange membrane, prevent the electrode solution from polluting the buffer solution, ensure the stability and accuracy of the electrophoresis process, and separate various proteins, especially proteins with different isoelectric points. BRIEF DESCRIPTION OF DRAWINGS

[0014] Fig. 1 It is the structural schematic diagram of the utility model;

[0015] Fig. 2 It is the structural schematic diagram of the utility model's electrophoresis separation chamber;

[0016] Fig. 3 It is the structural schematic diagram of the utility model.

[0017] In the figure, 1-cold liquid plate, 2-silica gel heat-conducting pad, 3-electrophoresis separation chamber, 31-separation chamber lower plate, 32-separation chamber upper plate, 33-electrode chamber, 34-carbon rod, 35-anion exchange membrane, 36-cation exchange membrane, 37-silica gel gasket, 38-separation chamber liquid inlet, 39-separation chamber liquid outlet, 4-round hole, 5-low-temperature cooling circulating pump, 6-electrode solution container, 7-electrode solution driving pump, 8-external electrophoresis instrument power supply, 9-gas-liquid buffer chamber, 10-sample driving pump, 11-protein sample. DETAILED DESCRIPTION

[0018] In order to make the personnel in the technical field better understand the utility model scheme, the technical scheme in the embodiment of the utility model will be described clearly and completely in the embodiment of the utility model below, obviously, the described embodiment is only a part of the embodiment of the utility model, not all. Based on the embodiment in the utility model, all other embodiments obtained by the ordinary skill in the art without making creative labor should belong to the scope of the utility model protection.

[0019] In the description of the utility model, it is understood that the orientation or position relation indicated by the terms "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like is the orientation or position relation shown based on the drawings, which is only for the convenience of describing the utility model and simplifying the description, and does not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the utility model.

[0020] Referring to Figs. 1-3 As shown in the figure, a protein free flow isoelectric focusing electrophoresis separation device, including cold liquid plate 1, the top of the cold liquid plate 1 is equipped with silica gel heat-conducting pad 2, the top of the silica gel heat-conducting pad 2 is provided with electrophoresis separation chamber 3, the electrophoresis separation chamber 3 includes separation chamber lower plate 31, the separation chamber lower plate 31 is connected with the silica gel heat-conducting pad 2, the top of the separation chamber lower plate 31 is provided with separation chamber upper plate 32, the top of the separation chamber upper plate 32 is connected with a pair of symmetrical electrode chamber 33, the electrode chamber 33 is connected with carbon rod 34, the bottom of the electrode chamber 33 is provided with the same size rectangular hole in the top of the separation chamber upper plate 32, the bottom of the separation chamber upper plate 32 is respectively bonded with anion exchange membrane 35 and cation exchange membrane 36 for isolating electrode liquid and running buffer solution at the corresponding position of rectangular hole, the separation chamber upper plate 32 and the separation chamber lower plate 31 are provided with silica gel gasket 37, the separation chamber upper plate 32 and the separation chamber lower plate 31 are connected through the clamp, the short side of the separation chamber upper plate 32 is respectively provided with a plurality of separation chamber liquid inlet 38 and separation chamber liquid outlet 39 on both sides.

[0021] The cold liquid plate 1 is circumscribed with low-temperature cooling circulating pump 5, which effectively maintains the temperature of the cold liquid plate, ensures that the temperature is always in the appropriate range during the whole separation process, helps to optimize the protein separation process, and avoids sample degradation caused by heat accumulation.

[0022] The top of the electrode chamber 33 is provided with a circular hole 4 for electrode liquid circulation on both sides, the electrode liquid container 6 and the electrode liquid driving pump 7 are connected through the pipeline between the two circular holes 4, one end of the carbon rod 34 above the anion exchange membrane 35 is connected with the negative electrode of the external electrophoresis instrument power supply 8, and one end of the carbon rod 34 above the cation exchange membrane 36 is connected with the positive electrode of the external electrophoresis instrument power supply 8, so that the electrode liquid can circulate in the electrode chamber, avoiding the change of the concentration and pH value of the electrode liquid, helping to stabilize the electric field, improving the efficiency and accuracy of electrophoresis separation, and accurately controlling the electric field strength and the acidity and alkalinity of the electrode liquid in the electrophoresis process, ensuring efficient separation under different experimental conditions.

[0023] The liquid inlet 38 of the separation chamber is connected with a gas-liquid buffer chamber 9 through a pipeline, the gas-liquid buffer chamber 9 is filled with a background buffer solution, the liquid outlet 39 of the separation chamber is connected with a collection device through a pipeline, the background buffer solution can stably enter the separation chamber 3, and the stability of the electrophoresis buffer is maintained. The liquid outlet 39 of the separation chamber is connected with the collection device, so that the separated sample can be efficiently collected and processed.

[0024] The electrode liquid container 6 which is in circulation exchange with the electrode chamber 33 above the anion exchange membrane 35 is filled with an alkaline solution, and the electrode liquid container 6 which is in circulation exchange with the electrode chamber 33 above the cation exchange membrane 36 is filled with an acidic solution. The alkaline and acidic electrode liquid containers are arranged according to the positions of the anion and cation exchange membranes, so that the pH value of the electrode liquid can be effectively controlled during the separation process, avoiding the influence of pH fluctuation on the electrophoresis result. Through independent circulation exchange of the acidic and alkaline electrode liquids, the electric field strength and stability are ensured, thereby improving the separation effect of the protein. Especially when dealing with proteins with different pI values, the resolution of the separation is higher.

[0025] The thicknesses of the silica gel gasket 37, the anion exchange membrane 35 and the cation exchange membrane 36 are all 1mm, which helps to improve the sealing performance of the device, prevents liquid leakage, ensures that no accidents occur during operation, and improves the safety of the experiment.

[0026] The material of the lower plate 31 of the separation chamber is glass, and the material of the upper plate 32 of the separation chamber is acrylic, which ensures transparent observation during the electrophoresis process and has sufficient durability.

[0027] One of the liquid inlets 38 of the separation chamber is connected with the protein sample 11 to be separated through a sample driving pump 10, so that the addition of the sample can be accurately controlled, improving the separation efficiency of the sample and the controllability of the experiment.

[0028] Working principle: The background buffer solution fills the gas-liquid buffer chamber 9 and enters the electrophoresis separation chamber 3 through the separation chamber inlet 38, the background buffer solution is a phosphoric acid buffer solution, the background solution provides a suitable ion strength and pH environment in the electric field, which helps the migration and focusing of the protein, the low-temperature cooling circulating pump 5 is started to ensure the circulation of the cooling liquid and reach the set low-temperature range, the sample driving pump 10 is started to add the protein sample 11 to be separated into the electrophoresis separation chamber 3 through the separation chamber inlet 38, the appropriate acidic solution or alkaline solution is added into the two electrode liquid containers 6 to ensure that the pH value of the liquid on both sides of the electrode chamber meets the experimental requirements, the electrode liquid driving pump 7 is started to ensure the circulation of the electrode liquid in the electrode chamber and stabilize the formation of the electric field, the negative electrode of the external electrophoresis instrument is connected to the carbon rod 34 on the anion exchange membrane 35, and the positive electrode is connected to the carbon rod 34 on the cation exchange membrane 36, according to the experimental requirements, appropriate voltage and current are set, the external electrophoresis instrument power supply 8 is started, and the electric field is uniformly distributed in the electrophoresis separation chamber 3, when the electric field acts on the electrophoresis separation chamber 3, the charge of the protein is opposite to the direction of the electric field, and the proteins with positive and negative charges will move to the negative and positive electrodes respectively, and the proteins will migrate along the pH gradient direction according to the isoelectric point (pI) in the electric field, due to the difference in pH of the background buffer solution and the electrode liquid in the solution, the proteins will migrate in different pH value regions under the action of the electric field, and finally focus at the isoelectric point position, at this time, the net charge of the protein is zero, the electrophoresis is stopped, and thus the separation is realized, with the continuous action of the electric field, the proteins with different isoelectric points will be separated into different bands and arranged along the electrophoresis direction in the separation chamber. The anion exchange membrane 35 and the cation exchange membrane 36 serve as key components in the electrophoresis separation system, which isolate the electrode liquid and the buffer solution, prevent the electrode liquid from polluting the electrophoresis buffer solution, and ensure the uniformity of the electric field and the stability of the electrophoresis process. The silica gel gasket 37 plays a sealing and isolating role between the upper and lower plates of the separation chamber, prevents solution leakage, and helps maintain the stability of the entire system. Since the protein separation is very sensitive to temperature, the cooling system (cooling liquid plate and circulating pump) in the device always maintains a low temperature to ensure that the protein does not denature or degrade during the separation process. After the electrophoresis separation is completed, the outlet 39 of the separation chamber is connected to the collection device, and the proteins focused at different isoelectric points can be collected.

[0029] It is obvious for those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application. Any reference signs in the claims should not be regarded as limiting the claims involved.

[0030] Furthermore, it should be understood that although the specification is described in terms of embodiments, not every embodiment includes only one independent technical solution, and the specification is described in this way only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A protein free-flow isoelectric focusing electrophoretic separation device comprising a cooling liquid plate (1), characterised in that: The cold liquid plate (1) is provided with silica gel heat-conducting pads (2) on the top, and an electrophoresis separation chamber (3) is arranged on the top of the silica gel heat-conducting pads (2), the electrophoresis separation chamber (3) comprises a separation chamber lower plate (31) connected with the silica gel heat-conducting pads (2), a separation chamber upper plate (32) is arranged above the separation chamber lower plate (31), a pair of symmetrical electrode chambers (33) are connected on the top of the separation chamber upper plate (32), carbon rods (34) are penetrated and connected in the electrode chambers (33), rectangular holes with the same size are formed in the bottom of the electrode chambers (33) and the top of the separation chamber upper plate (32), anion exchange membranes (35) and cation exchange membranes (36) for isolating electrode liquid and running buffer solution are respectively adhered to the bottom of the separation chamber upper plate (32) at positions corresponding to the rectangular holes, silica gel gaskets (37) are arranged between the separation chamber upper plate (32) and the separation chamber lower plate (31), the separation chamber upper plate (32) and the separation chamber lower plate (31) are connected through a clamp, a plurality of separation chamber liquid inlets (38) and separation chamber liquid outlets (39) are respectively formed in the two sides of the short side of the separation chamber upper plate (32).

2. A protein free-flow isoelectric focusing electrophoresis separation device according to claim 1, characterized in that: The cold liquid plate (1) is externally connected with a low-temperature cooling circulating pump (5).

3. A protein free-flow isoelectric focusing electrophoresis separation device according to claim 2, characterized in that: Round holes (4) for electrode liquid circulation are formed in the top of the electrode chambers (33) on both sides, an electrode liquid container (6) and an electrode liquid driving pump (7) are connected through pipelines between the two round holes (4), one end of the carbon rod (34) above the anion exchange membrane (35) is connected with a negative electrode of an external electrophoresis instrument power supply (8), and one end of the carbon rod (34) above the cation exchange membrane (36) is connected with a positive electrode of the external electrophoresis instrument power supply (8).

4. A protein free-flow isoelectric focusing electrophoresis separation device according to claim 3, characterized in that: The separation chamber liquid inlets (38) are connected with gas-liquid buffer chambers (9) through pipelines, the gas-liquid buffer chambers (9) are filled with background buffer solution, and the separation chamber liquid outlets (39) are connected with a collection device through pipelines.

5. A protein free-flow isoelectric focusing electrophoresis separation device according to claim 4, characterized in that: The electrode liquid container (6) in circulation exchange with the electrode chamber (33) above the anion exchange membrane (35) is filled with alkaline solution, and the electrode liquid container (6) in circulation exchange with the electrode chamber (33) above the cation exchange membrane (36) is filled with acidic solution.

6. A protein free-flow isoelectric focusing electrophoretic separation device according to claim 5, characterized in that: The thicknesses of the silica gel gaskets (37), the anion exchange membranes (35) and the cation exchange membranes (36) are all 1 mm.

7. A protein free-flow isoelectric focusing electrophoretic separation device according to claim 6, characterized in that: The separation chamber lower plate (31) is made of glass, and the separation chamber upper plate (32) is made of acrylic.

8. A protein free-flow isoelectric focusing electrophoretic separation device according to claim 7, characterized in that: One of the separation chamber liquid inlets (38) is connected with a protein sample (11) to be separated through a sample driving pump (10).

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