CN109270153B - Non-amphoteric electrolyte free-flow isoelectric focusing electrophoresis separation method - Google Patents
Non-amphoteric electrolyte free-flow isoelectric focusing electrophoresis separation method Download PDFInfo
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- CN109270153B CN109270153B CN201811429548.2A CN201811429548A CN109270153B CN 109270153 B CN109270153 B CN 109270153B CN 201811429548 A CN201811429548 A CN 201811429548A CN 109270153 B CN109270153 B CN 109270153B
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Abstract
The application discloses a non-ampholyte free-flow isoelectric focusing electrophoresis separation method, which comprises the following steps: pumping the running mobile phase into a separation cavity of the free flow electrophoresis device by using a driving pump, and keeping the mobile phase running stably; the second step is that: pumping positive electrode liquid in an electrode liquid storage tank into a positive electrode chamber of the free flow electrophoresis device by using an electrode liquid circulating pump and keeping the electrode liquid circulating, and pumping negative electrode liquid in the electrode liquid storage tank into a negative electrode chamber of the free flow electrophoresis device and keeping the electrode liquid circulating; the third step: applying voltage to the two electrodes, adjusting the voltage to proper value, and keeping the device running stably, so that the device automatically forms a stable pH gradient between the electrodes at the rear part of the separation cavity. The application has the advantages that: the method is simple and easy to implement, the required reagent is low in price, and the detection of the separated sample cannot be influenced.
Description
Technical Field
The invention relates to the technical field of free flow electrophoresis separation, in particular to an isoelectric focusing electrophoresis separation method of free flow without amphoteric electrolyte.
Background
Proteins are the material basis of life activities, and are various in variety and function. It is a biological macromolecule which is formed by connecting amino acids through amido bonds and has a specific structure and function. It can perform various biological functions, enabling the information contained in DNA to be revealed. Proteins are the most direct embodiment of life activities, and only proteome studies can fundamentally explain life processes. The technology of protein isolation is the basis of proteome research. However, due to the specific properties of the protein itself, it is difficult to perform efficient separation using conventional separation techniques.
Free flow electrophoresis is a continuous full liquid phase separation technology, and the device comprises a thin separation cavity formed by two parallel plates which are closely spaced. The mobile phase is operated to flow through the separation chamber to form a stable laminar flow. After entering the separation cavity through the sample inlet, the sample is driven by liquid flow to the outlet end. Meanwhile, an orthogonal transverse electric field is loaded on the laminar flow of the liquid flowing longitudinally in the device, so that the charged sample in the laminar flow is transversely deviated to different degrees according to different electrophoretic mobility under the action of the electric field force, and the separation effect is achieved. The sample continuously enters in the free flow electrophoresis separation process, has no solid supporting medium, has mild separation environment, and is particularly suitable for separation and preparation of biological samples such as protein and the like. The Cao-becoming-popular subject group develops an easy-to-operate stable free-flow electrophoresis device with a cooling system in 2013, and the operability of the free-flow electrophoresis device is greatly improved. Free flow electrophoresis can be combined with other electrophoresis technologies, and the advantages of the other electrophoresis technologies are fully utilized, so that the free flow electrophoresis can play a role in more fields. The most common separation modes are free-flow zone electrophoresis, free-flow isoelectric focusing, free-flow isotachophoresis, free-flow electric field gradient electrophoresis, and the like. The Cao-Happy topic group developed a mobile reaction interface free flow electrophoresis method in 2017 for continuous concentration of proteins. Free-flow isoelectric focusing electrophoresis has the highest resolution among the common free-flow electrophoresis separation modes and is therefore often used for separation of samples that are difficult to separate. The Cao-favored subject group successfully prepared endogenous mitochondria in 2017 by using a circulating free current isoelectric focusing device. The method uses a series of high-concentration ampholytes with pH gradient as a running mobile phase, and establishes the pH gradient in a circulating free current isoelectric focusing device to separate samples. Isoelectric focusing was a major means for protein separation that emerged in the 60's of the 20 th century. It has been rapidly developed because of its higher resolution. The basic principle is to separate and analyze proteins in a continuous linear pH gradient by using different isoelectric points of various protein samples. The isoelectric focusing process is a sample focusing process, so that the problem of band diffusion is small, and the separation and concentration can be simultaneously achieved. In conventional free-flow isoelectric focusing electrophoresis, a specific ampholyte buffer solution is required to establish the pH gradient. Ampholyte solutions, however, are expensive and may interact with portions of the sample, affecting the sensitivity of the uv detection. When the separated components are to be detected by mass spectrometry, these ampholytes must be removed by complicated means. Therefore, free-flow isoelectric focusing electrophoresis without using ampholyte buffer solution would be an ideal method of operation.
Disclosure of Invention
In order to overcome the defects of the prior art, the application provides a non-ampholyte free-flow isoelectric focusing electrophoresis separation method.
The technical scheme adopted by the application is as follows:
a non-amphoteric electrolyte free flow isoelectric focusing electrophoresis separation method comprises the following specific steps:
the first step is as follows: pumping the running mobile phase into a separation cavity of the free flow electrophoresis device by using a driving pump, and keeping the mobile phase running stably;
the second step is that: pumping positive electrode liquid in an electrode liquid storage tank into a positive electrode chamber of the free flow electrophoresis device by using an electrode liquid circulating pump and keeping the electrode liquid circulating, and pumping negative electrode liquid in the electrode liquid storage tank into a negative electrode chamber of the free flow electrophoresis device and keeping the electrode liquid circulating;
the third step: applying voltage to the two electrodes, adjusting the voltage to proper value, and keeping the device running stably, so that the device automatically forms a stable pH gradient between the electrodes at the rear part of the separation cavity.
The free flow isoelectric focusing electrophoresis method uses a free flow electrophoresis device which comprises a running mobile phase liquid storage bottle, a running mobile phase driving pump, a gas-liquid buffer device, a free flow electrophoresis cavity, a positive electrode chamber, a negative electrode chamber, a positive electrode liquid circulating pump, a negative electrode liquid circulating pump, a positive electrode liquid storage bottle, a negative electrode liquid storage bottle, a high-voltage power supply and a component collecting device; the running mobile phase liquid storage bottle is connected with the running mobile phase driving pump through a hose, the running mobile phase driving pump is connected with a gas-liquid buffering device through a hose, the gas-liquid buffering device is connected with a free flow electrophoresis cavity through a hose, the free flow electrophoresis cavity is connected with a component collecting device through a hose, the free flow electrophoresis cavity is connected with a positive electrode chamber through a cation exchange membrane, the positive electrode chamber is connected with a high-voltage power supply positive electrode through an electrode rod, the positive electrode chamber is connected with a positive electrode liquid circulating pump through a hose, the positive electrode liquid circulating pump is connected with the positive electrode liquid storage bottle through a hose, the free flow electrophoresis cavity is connected with a negative electrode chamber through an anion exchange membrane, the negative electrode chamber is connected with a high-voltage power supply negative electrode through an electrode rod, the negative electrode chamber is connected with a negative electrode liquid circulating pump through a hose, and the negative electrode liquid circulating pump is connected with the negative electrode liquid storage bottle through a hose.
The anode of the free-flow electrophoresis device uses acidic electrode liquid, and the concentration of the acidic electrode liquid is 0.01-1 mol/L.
The cathode of the free-flow electrophoresis device uses alkaline electrode liquid, and the concentration of the alkaline electrode liquid is 0.01-1 mol/L.
The free flow electrophoresis device uses a buffer solution as an operation mobile phase during operation, and the concentration of the buffer solution is 0.001mol/L-0.1 mol/L.
The flow rate of the mobile phase in the free flow electrophoresis device is 1ml/min-100 ml/min.
The free-flow isoelectric focusing electrophoresis method can form a pH gradient of 2-10, preferably 3-8, under the action of electric field force.
The application provides an application of free-flow isoelectric focusing electrophoresis without ampholyte in protein separation.
Compared with the prior art, the invention has the beneficial effects that:
the pH gradient is formed without the use of expensive special ampholytes as the mobile phase of operation. The hydrogen ions and hydroxyl ions in the electrode liquid in the electrode chamber can automatically form pH gradient in the free flow electrophoresis chamber under the action of electric field force.
The anode liquid in the anode electrode chamber is recycled with the anode liquid in the anode liquid storage bottle through an anode liquid circulating pump, so that the concentration of the anode liquid in the anode electrode chamber is kept stable. The negative electrode solution in the negative electrode chamber is recycled with the negative electrode solution in the negative electrode solution storage bottle through the negative electrode solution circulating pump, so that the concentration of the negative electrode solution in the negative electrode chamber is kept stable. Thereby ensuring the stability of the pH gradient in the free flow electrophoresis chamber.
The buffer solution used does not interact with the sample, increasing the sensitivity of the UV detection relative to ampholytes as the mobile phase of operation.
When the components separated by free flow electrophoresis need to be connected with mass spectrometry detection, the ion current cannot be increased and desalting treatment is not needed due to the low concentration of the used buffer solution.
Drawings
Fig. 1 is a schematic diagram of a free-flow electrophoresis apparatus.
Fig. 2 is a schematic diagram of a free-flow electrophoresis separation chamber.
FIG. 3 is a pH gradient curve;
FIG. 4 is a mass spectrogram of an egg white protein isolate detected by a matrix-assisted laser desorption ionization source time-of-flight mass spectrometer; fig. 4a is a schematic view of a first hole, fig. 4b is a schematic view of a fourth hole, and fig. 4c is a schematic view of a ninth hole.
The labels in the figures are: 1-running a mobile phase driving pump, 2-a negative electrode liquid circulating pump, 3-a positive electrode liquid circulating pump, 4-a sample injection pump, 5-a negative electrode liquid storage bottle, 6-running a mobile phase liquid storage bottle, 7-a sample solution storage bottle, 8-a positive electrode liquid storage bottle, 9-a high-voltage power supply, 10-an air cylinder, 11-an air-liquid buffer device, 12-a free flow electrophoresis cavity and 13-a component collecting device; 121-positive electrode chamber, 122-negative electrode chamber, 123-free flow electrophoresis separation chamber, 124-running mobile phase input pipeline.
Detailed Description
The following detailed description of the invention and its preferred embodiments are provided to illustrate but not to limit the invention, and are provided in the accompanying drawings.
Example 1
Referring to fig. 1, a schematic diagram of a free flow electrophoresis apparatus used in the present application is shown. The device comprises: an operation mobile phase driving pump (1) is used for driving the operation mobile phase in the operation mobile phase liquid storage bottle (6) to enter the gas-liquid buffer device (11). Removing air bubbles in the running mobile phase by using an air cylinder (10) in an air-liquid buffer device (11) so that the running mobile phase is filled in a free flow electrophoresis cavity (12); the negative electrode liquid circulating pump (2) is used for driving the negative electrode liquid circulation in the negative electrode liquid storage tank (5), and the positive electrode liquid circulating pump (3) is used for driving the positive electrode liquid circulation in the positive electrode liquid storage tank (8); the sample pump (4) is used for pumping the sample in the sample solution storage bottle (7) into the free flow electrophoresis cavity (12); a high voltage power supply (9) for providing a device voltage; a component collection device (13).
Referring to fig. 2, a schematic diagram of a free-flow electrophoresis chamber of a free-flow electrophoresis apparatus used in the present invention is shown. The device comprises: an operating mobile phase input conduit (124); a positive electrode chamber (121) is separated from the free flow electrophoresis separation cavity (123) by a cation exchange membrane; a negative electrode chamber (122) is separated from the free flow electrophoresis separation chamber (123) by an anion exchange membrane.
In the process of free flow isoelectric focusing electrophoresis, in the running direction of the running mobile phase, the running mobile phase drives the pump (1) to provide driving force, so that the running mobile phase and the sample solution flow from the inlet to the outlet of the free flow electrophoresis chamber (12). The direction perpendicular to the running mobile phase can be regarded as a process of isoelectric focusing. In the process, the high-voltage power supply (9) provides voltage, so that hydrogen ions in the positive electrode chamber (121) move to the negative electrode through the cation exchange membrane of the positive electrode chamber (121) under the action of an electric field, and hydroxyl ions in the negative electrode chamber (122) move to the positive electrode through the anion exchange membrane of the negative electrode chamber (122) under the action of the electric field. The operating mobile phase near the positive electrode is acidic due to the increase in hydrogen ion concentration, while the operating mobile phase near the negative electrode is alkaline due to the increase in hydroxide ion concentration. In the middle part of the free flow electrophoresis chamber (12), a nearly linear pH value gradient is formed due to the migration of hydrogen ions and hydroxyl ions under the action of an electric field, and the pH value gradient curve is shown in figure 3.
Example 2
In the first step, an operating mobile phase driving pump (1) is opened to pump the operating mobile phase in an operating mobile phase liquid storage bottle (6) into a gas-liquid buffer device (11). Air bubbles in the running mobile phase are removed by using an air cylinder (10) in an air-liquid buffer device (11), so that the running mobile phase is filled in a free flow electrophoresis cavity (12) and the flow rate is controlled to be 1 ml/min. And opening the negative electrode liquid circulating pump (2) and the positive electrode liquid circulating pump (3) to enable positive and negative electrode liquid in the negative electrode liquid storage tank (5) and the positive electrode liquid storage tank (8) to be pumped into positive and negative electrode chambers of the free flow electrophoresis chamber, and keeping the electrode liquid circulating. And turning on a high-voltage power supply (9) and regulating to 400V voltage. Keeping the device running stable for 30min to make it stable. And opening the sample pump (4) to pump the egg white sample solution in the sample solution storage bottle (7) into the free flow electrophoresis chamber (12) at the speed of 1 ml/min. After running for 1h under the action of the electric field force, the sample is separated and concentrated and then collected by a component collecting device (13). The collected egg white protein separation component was detected by a matrix-assisted laser desorption ionization source time-of-flight mass spectrometer, and the results obtained are shown in fig. 4: by using the free flow isoelectric focusing electrophoresis method, three protein components in the egg white solution are completely separated according to the isoelectric points. And desalting treatment is not needed before mass spectrum detection.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the concept of the present invention, and these modifications and decorations should also be regarded as being within the protection scope of the present invention.
Claims (1)
1. A non-amphoteric electrolyte free flow isoelectric focusing electrophoresis separation method is characterized by comprising the following specific steps:
the first step is as follows: pumping the running mobile phase into a separation cavity of the free flow electrophoresis device by using a driving pump, and keeping the mobile phase running stably;
the second step is that: pumping positive electrode liquid in an electrode liquid storage tank into a positive electrode chamber of the free flow electrophoresis device by using an electrode liquid circulating pump and keeping the electrode liquid circulating, and pumping negative electrode liquid in the electrode liquid storage tank into a negative electrode chamber of the free flow electrophoresis device and keeping the electrode liquid circulating;
the third step: applying voltage to the two electrodes, adjusting the voltage to a proper value, and keeping the device stably running to ensure that the device automatically forms a stable pH gradient between the electrodes at the rear part of the separation cavity;
the pH gradient automatically formed between the electrodes is along the transverse direction of the separation cavity, the running mobile phase near the anode is acidic due to the increase of the concentration of hydrogen ions, the running mobile phase near the cathode is alkaline due to the increase of the concentration of hydroxyl ions, and a nearly linear pH value gradient is formed between the running mobile phase and the cathode due to the migration of the hydrogen ions and the hydroxyl ions under the action of an electric field;
the running mobile phase is a buffer solution, wherein the salt concentration is 0.001-0.1 mol/L, and the pH value is 2-12;
the anode electrode liquid is HCl acid solution, the pH value range is 2-7, and the concentration is 0.01-1 mol/L;
the negative electrode solution is NaOH alkaline solution, the pH value range is 7-12, and the concentration is 0.01-1 mol/L;
the rear part of the separation cavity refers to the position of 50-70% of the longitudinal direction of the separation cavity; the pH gradient ranges from 2 to 10.
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| CN104056468A (en) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | Electrofocusing method for separating amphoteric matter without amphoteric electrolyte |
| CN104307367A (en) * | 2014-10-27 | 2015-01-28 | 上海交通大学 | Reciprocating type free flow isoelectric focusing electrophoresis device and method |
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| CN104056468A (en) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | Electrofocusing method for separating amphoteric matter without amphoteric electrolyte |
| CN104307367A (en) * | 2014-10-27 | 2015-01-28 | 上海交通大学 | Reciprocating type free flow isoelectric focusing electrophoresis device and method |
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| Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins;Song-Fang Xie et.al;《Journal of Separation Science》;20180531;第41卷(第9期);第2085-2089页 * |
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