1. A method for extracting the vacuolar protein of the fruit cells of woody plants apples is characterized by comprising the following steps:

(1) taking apple fruits as experimental test materials, and extracting protoplasts from apple pulp cells;

(2) separating the protoplast obtained in the step (1) to obtain vacuoles;

(3) carrying out ultralow-temperature freeze thawing treatment on the vacuole obtained in the step (2) to obtain apple fruit cell vacuole protein;

(4) and (4) carrying out SDS polyacrylamide gel electrophoresis detection on the vacuole protein obtained in the step (3), and finally determining to obtain the apple fruit cell vacuole protein.

2. The protoplast extraction method according to claim 1, wherein in step (1), the apple pulp is added into a sterilized culture dish and crushed, and then the treated cell wall enzymolysis liquid is added, mixed evenly, and processed in the dark, and then placed on an open-air shaking table for enzymolysis for 2-3 hours at room temperature; and (3) adding a W5 buffer solution after the enzymolysis is finished, uniformly mixing, filtering by using a nylon gauze, and standing or centrifuging the filtered material at room temperature to obtain a precipitate, namely the protoplast.

3. The extraction method according to claim 2, wherein the cell wall enzymolysis solution is prepared by mixing 4ml of mannitol (1M), 200. mu.l of KCl (1M), 402. mu.l MES (0.5M), 0.2g of sucrose, 0.15g of MESCellulase R-10, 0.04g of Segrenase R-10, 0.005g of pectinase Y-23 and ddH₂O is obtained by metering the volume to 10 ml; the treatment comprises the following steps: heating the prepared cell wall enzymolysis liquid at 55 deg.C for 10min, cooling the cell wall enzymolysis liquid at 0 deg.C for 5min, and adding 100 μ l CaCl₂(1M)。

4. The extraction method according to claim 2, wherein the W5 buffer solution is prepared by mixing 6.16ml NaCl (5M), 25ml CaCl₂ddH for (1M), 1ml KCl (1M), 0.8ml MES (0.5M)₂And O is obtained by metering to 200 ml.

5. The extraction method according to claim 1, wherein in the step (2), the separation method comprises: adding a lysis buffer solution into the protoplast obtained in the step (1), uniformly mixing, and then standing at room temperature; adding 4% of polysucrose solution, adding vacuole buffer solution, and centrifuging; after centrifugation, separating and subpackaging each centrifugal layer to obtain vacuoles; the 4% ficoll solution is obtained by mixing a vacuole buffer solution and a lysis buffer solution.

6. The extraction method according to claim 5, wherein the lysis buffer is prepared by adding 3ml of mannitol (1M), 5ml of 30% (wt) polysucrose, 300. mu.l of EDTA (0.5M), 375. mu.l of first sodium phosphate buffer, 75. mu.l of 0.1% neutral red solution in ddH₂O is obtained by fixing the volume to 15 ml; the lysis buffer was stored in a 37 ℃ water bath.

7. The extraction method according to claim 5, wherein the vacuolar buffer is prepared by adding 4.5ml of mannitol (1M), 250. mu.l of sodium diphosphate buffer, 40. mu.l of EDTA (0.5M) with ddH₂O is obtained by metering the volume to 10 ml; the vacuolar buffer was stored on ice.

8. The extraction method according to claim 1, wherein in the step (3), the ultra-low temperature freezing and thawing method is: freezing the vacuole obtained in the step (2) at-80 ℃ overnight, and freezing and crushing the vacuole to obtain vacuole protein.

9. The extraction method according to claim 1, wherein in the step (4), the SDS polyacrylamide gel electrophoresis detection method comprises: taking out the vacuole protein obtained in the step (3), placing the vacuole protein on ice for melting, adding the sample buffer solution after the vacuole protein is completely melted, fully and uniformly mixing, boiling the mixture in boiling water, and carrying out SDS polyacrylamide gel electrophoresis detection after the boiling is finished.

10. The detection method according to claim 9, wherein the voltage is adjusted to 110V before running the gel to the interface between the concentrated gel and the separated gel and 130V when the running gel reaches the interface, when SDS polyacrylamide gel electrophoresis is used.

Background

Vacuoles are organelles consisting of a single membrane and the cellular fluid inside, a thin membrane at the place where the cytoplasm is separated from the vacuole is called the vacuole membrane, the composition and the characteristics of the membrane are the same as those of the cytoplasmic membrane, and the fluid in the vacuole is called the cellular fluid. The plant vacuole is a multifunctional organelle, has various physiological functions of participating in permeation regulation, storing organic metabolites, regulating pH and the like, and participates in various physiological processes of plant cell water physiology, nutrition physiology, photosynthetic physiology, growth physiology, reproductive physiology, environmental physiology and the like. It is not certain that vacuoles are a critical presence in plant cells, and therefore, the study of vacuoles is essential. While the research on vacuole protein has been a hot spot for studying the vacuole function of plants, the structural and physiological complexity of vacuoles makes the relevant research on vacuoles very challenging.

It is known that in herbaceous plants, it has been possible to successfully isolate a large number of high-purity vacuoles from the root tips of tomato seedlings, red beet root storage tissue, halogetic tobacco leaves, Arabidopsis cell suspensions, and Arabidopsis rosette leaves, in which Arabidopsis is well studied in this respect, and to identify, by proteomic analysis, a variety of tonoplast proteins including tonoplast proteins with good properties, such as type V H⁺ATPases and form V H⁺-PPases, and other vacuolar membrane-associated but functionally unknown proteins; in woody plants, few studies on the extraction of vacuolar proteins have been made, and some of them have been in the stage of protoplast extraction, and it is known that a large amount of high-purity vacuoles can be successfully isolated from the leaves of the vinca rosea, a shrub plant, but the studies on the extraction of vacuolar proteins have not been made yet. The lignification degree of the cell walls of the apple serving as a perennial deciduous tree is far greater than that of the woody plant and the subshrub plant, the cell walls are thicker and harder as the lignification degree is higher, great difficulty exists in the stage of extracting the protoplast, the dosage and enzymolysis time of various enzymes are required to be researched, and the vacuole protein can be further researched after a large amount of complete protoplast is ensured to be extracted. Therefore, the extraction of vacuolar proteins from apple-related tissues is undoubtedly a huge innovation and challenge, requiring repeated research and experimentation in each step, whether the extraction of protoplasts, and thus vacuoles, and finally vacuolar proteins.

Disclosure of Invention

In view of the above prior art, the present invention aims to provide a method for extracting vacuolar protein from fruit cells of a woody plant apple, by which vacuolar protein in apple fruits can be efficiently obtained.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for extracting a woody plant apple fruit cell vacuole protein, which comprises the following steps:

(1) selecting good-state apple fruits as experimental test materials, and extracting protoplasts from apple pulp cells;

(2) separating the protoplast obtained in the step (1) to obtain vacuoles;

(3) carrying out ultralow-temperature freeze thawing treatment on the vacuole obtained in the step (2) to obtain apple fruit cell vacuole protein;

(4) and (4) carrying out SDS polyacrylamide gel electrophoresis detection on the vacuole protein obtained in the step (3), and finally determining to obtain the apple fruit cell vacuole protein.

Preferably, in step (1), the method for extracting protoplasts comprises: adding the apple pulp in a good state into a sterilized culture dish, grinding, adding the treated cell wall enzymolysis liquid, mixing uniformly, processing in a dark place, placing on an open-air shaking table, and performing enzymolysis for 2-3 hours at room temperature; and (3) adding a W5 buffer solution after the enzymolysis is finished, uniformly mixing, filtering by using a nylon gauze, and standing or centrifuging the filtered material at room temperature to obtain a precipitate, namely the protoplast.

Preferably, the cell wall enzymolysis solution is prepared by adding 4ml of mannitol (1M), 200. mu.l of KCl (1M), 402. mu.l of MES (0.5M), 0.2g of sucrose, 0.15g of cellulase R-10, 0.04g of macerase R-10, 0.005g of pectinase Y-23 and ddH₂O is obtained by metering the volume to 10 ml; the treatment comprises the following steps: heating the prepared cell wall enzymolysis liquid at 55 deg.C for 10min, cooling the cell wall enzymolysis liquid at 0 deg.C for 5min, and adding 100 μ l CaCl₂(1M)。

Preferably, the W5 buffer solution is prepared by mixing 6.16ml NaCl (5M), 25ml CaCl₂(1M)、1ml KCl(1M) 0.8ml of DDH for MES (0.5M)₂And O is obtained by metering to 200 ml.

Preferably, in step (2), the separation to obtain vacuoles is performed by: adding a lysis buffer solution into the protoplast obtained in the step (1), uniformly mixing, and then standing at room temperature; adding 4% of polysucrose solution, adding vacuole buffer solution, and centrifuging; after centrifugation, separating and subpackaging each centrifugal layer, and determining the centrifugal layer containing a large amount of vacuoles through an optical microscope to obtain vacuoles; the 4% ficoll solution is obtained by mixing a vacuole buffer and a lysis buffer.

Preferably, the lysis buffer is prepared by adding 3ml of mannitol (1M), 5ml of 30% (wt) polysucrose, 300. mu.l EDTA (0.5M), 375. mu.l of first sodium phosphate buffer, 75. mu.l of 0.1% neutral red solution in ddH₂O is obtained by fixing the volume to 15 ml; the lysis buffer was incubated in a 37 ℃ water bath.

Preferably, the vacuole buffer is prepared by adding 4.5ml of mannitol (1M), 250. mu.l of sodium diphosphate buffer, 40. mu.l of EDTA (0.5M) with ddH₂O is obtained by metering the volume to 10 ml; the vacuolar buffer was stored on ice.

Preferably, in the step (3), the ultra-low temperature freezing and thawing method comprises the following steps: freezing the vacuole obtained in the step (2) at-80 ℃ overnight, and freezing and crushing the vacuole to obtain vacuole protein.

Preferably, in step (4), the SDS polyacrylamide gel electrophoresis method is: taking out the vacuole protein obtained in the step (3), placing the vacuole protein on ice for melting, adding the sample buffer solution after the vacuole protein is completely melted, fully and uniformly mixing, boiling the mixture in boiling water, and carrying out SDS polyacrylamide gel electrophoresis detection after the boiling is finished.

Preferably, when SDS polyacrylamide gel electrophoresis is used, the voltage is adjusted to 110V before running the gel to the interface between the concentrated gel and the separated gel, and to 130V when the gel reaches the interface.

The invention has the beneficial effects that:

the invention extracts vacuole protein from apple pulp tissue through repeated research and test, the extraction method has high efficiency, and the vacuole extraction amount is about 100 per mm²Vacuolar protein extractionThe concentration was about 1. mu.g/ml.

Drawings

FIG. 1: (a) view of protoplasts under 10-fold mirror, (b) view of protoplasts under 40-fold mirror;

FIG. 2: (a) and (b) is a view of the vacuole under 10 times of the mirror, (c) and (d) is a view of the vacuole under 20 times of the mirror;

FIG. 3: and (4) detecting the vacuole protein of the apple fruit cell obtained by separation by SDS polyacrylamide gel electrophoresis.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background art, apple, which is a perennial deciduous tree, has a cell wall with a higher degree of lignification than other woody plants and sub-shrub plants, and has a much greater difficulty in the stage of extracting protoplasts, because the cell wall is thicker and harder as the degree of lignification is higher; on the other hand, vacuole is a single-layer membrane organelle, and is wrapped by protoplast, and other organelles exist between the cell membrane and the protoplast, so the fragility of vacuole and the complexity of the environment in which the vacuole is located are also a difficulty of experiments.

Based on the method, the invention provides a method for extracting the vacuolar protein of the fruit cells of the woody plant apple. In the invention, a large amount of high-purity protoplasts can be extracted by properly improving a laboratory protoplast extraction method and carrying out short-time enzymolysis and filtration precipitation. The vacuole extraction part of the invention utilizes a lysis solution containing high-concentration ficoll to enable protoplasts to generate osmotic shock and quickly release vacuoles, then the protoplasts are stored in ficoll solutions with different concentrations (from bottom to top, the lysis buffer solution containing 30% ficoll solution, the ficoll solution containing 4% ficoll solution and the vacuole buffer solution containing no ficoll solution are respectively stored in different temperatures and can not be mixed after being added, so that the protoplasts generate heat shock to promote the vacuole release and form a buffer process of vacuole release and collection, the ficoll is white powder, nontoxic and harmless), and ultracentrifugation is carried out to obtain vacuoles. The vacuole protein is obtained by breaking vacuoles and separating through an ultralow temperature freeze thawing method, and the method is simple, convenient and quick. In the invention, the vacuolar protein is detected by SDS-polyacrylamide gel electrophoresis, and compared with the existing SDS-PAGE gel electrophoresis, the detection method is different from the concentration of the prepared concentrated gel and separation gel, the magnitude of the adjusting voltage, the used dye solution (a SDS-PAGE protein gel rapid dye solution) and the like.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available.

Description of the drawings: the preparation method of the solution used in the embodiment comprises the following steps:

(1) mannitol (1M): 36.435g of mannitol solid are weighed out and finally ddH is used₂O is added to the volume of 200 ml.

(2)CaCl₂(1M): 22.196g of CaCl were weighed₂Solid, finally with ddH₂O is added to the volume of 200 ml.

(3) NaCl (5M): 58.44g NaCl solid are weighed out and finally ddH is used₂O is added to the volume of 200 ml.

(4) KCl (1M): 14.19g of KCl solid is weighed and finally ddH is used₂O is added to the volume of 200 ml.

(5)MgCl₂(1M): 40.66g of MgCl were weighed₂Solid, finally with ddH₂O is added to the volume of 200 ml.

(6) MES (0.5M): 21.32g of MES solid are weighed out and finally ddH is used₂O is added to the volume of 200 ml. (pH adjusted to 5.7)

(6) EDTA (0.5M): 74.45g of EDTA are weighed out and finally ddH is used₂O is added to the volume of 400 ml. (pH adjusted to 8.0 with solid NaOH)

(7)30％(wt) ficoll solution: 4.5g of ficoll are weighed out and finally ddH is used₂O is added to the volume of 15 ml.

(Note: the ficoll is very insoluble, can be dissolved by dividing into a plurality of times, can be dissolved by itself after being placed under the condition of 65 ℃ water bath for a short time and heated for a few minutes, can also be dissolved naturally at normal temperature from half a day to one day in advance, the heating temperature is not too high, the heating temperature is not too long, otherwise the vacuole cracking effect and the final separation and collection can be influenced.)

(8) 0.1% neutral red solution: 0.1g neutral red, 200. mu.l 1% acetic acid, 50. mu.l chloroform was added, followed by ddH₂O is added to 100 ml.

(9) Solution A: 6.24g NaH were weighed₂PO₄·2H₂O, finally with ddH₂O is added to the volume of 200 ml.

(10) Solution B: 14.333g of Na were weighed₂HPO₄·12H₂O, finally with ddH₂O is added to the volume of 200 ml.

(11) First sodium phosphate buffer (0.2M): 16ml of solution A and 84ml of solution B were mixed.

(12) Sodium diphosphate buffer (0.2M): 5.3ml of solution A and 94.7ml of solution B were mixed.

(13) Cell wall enzymolysis liquid (used in the first place): adding 4ml mannitol (1M), 200. mu.l KCl (1M), 402. mu.l MES (0.5M), 0.2g sucrose, 0.15g cellulase R-10, 0.04g macerase R-10, 0.005g pectinase Y-23 into a 50ml clean centrifuge tube with scales, and adding ddH₂The volume of O is adjusted to 10ml, and the solute is dissolved thoroughly by shaking lightly from time to time.

(14) W5 buffer (now ready for use): 6.16ml NaCl (5M), 25ml CaCl were added₂(1M), 1ml KCl (1M), 0.8ml MES (0.5M), finally with ddH₂O is added to the volume of 200 ml.

(15) MMG buffer (now ready for use): 80ml of mannitol (1M) and 3ml of MgCl were added₂(1M), 1.6ml MES (0.5M), finally with ddH₂O is added to the volume of 200 ml.

(16) Lysis buffer (now ready for use): 3ml mannitol (1M), 5ml 30% (wt) polysucrose, 300. mu.l EDTA (0.5M), 375. mu.l sodium diphosphate buffer, 75μ l of 0.1% neutral Red solution, finally with ddH₂And (4) metering the volume of O to 15ml, and preserving the heat in a water bath at 37 ℃.

(17) Vacuolar buffer (now ready for use): add 4.5ml mannitol (1M), 250. mu.l first sodium phosphate buffer, 40. mu.l EDTA (0.5M), finally with ddH₂And O is metered to 10ml and placed on ice for standby.

(18) 4% ficoll solution (now ready for use): 4.5ml of the vacuole buffer and 3ml of the lysis buffer were mixed and left at room temperature until use (about 25 ℃).

(19) Loading buffer solution: 625ml of 1.0M Tris-HCl (pH6.8), 2ml of glycerol, 2ml of 10% SDS, 1ml of beta-mercaptoethanol, 0.5ml of 0.1% bromophenol blue and finally ddH₂O is added to the volume of 10 ml.

(20) Gel buffer (1.5M, pH 8.8): 18.17g Tris were weighed and ddH was added₂O dissolution, pH adjustment to 8.8 with 6M HCl and finally ddH₂And O is metered to 100ml and stored at 4 ℃.

(21) Concentrated gel buffer (1.0M, pH 6.8): weigh 12.12g Tris, add ddH₂O dissolution, pH adjustment to 6.8 with 6M HCl and finally ddH₂And O is metered to 100ml and stored at 4 ℃.

(22) 30% gel stock (30% Acrylamide): 29.9g of acrylamide (Acr), 0.8g of methylenebisacrylamide (Bis) were weighed out and finally ddH was used₂And (4) fixing the volume of O to 100ml, wrapping the bottle body with tin foil paper, and storing at 4 ℃ for use within 30 days.

(23) Decoloring liquid: 50ml of methanol, 75ml of glacial acetic acid and finally 875ml of ddH are added₂And (4) mixing the materials.

(24) 10% SDS-PAGE separation gel formulation (amount of one gel made): add 1.9ml of ddH₂O, 1.7ml of 30% Acrylamide, 1.3ml of 1.5M Tris-HCl (pH8.8), 0.05ml of 10% SDS, 0.05ml of 10% (W/V) ammonium persulfate, 0.02ml of TEMED.

(25) Preparation of 5% Acrylamide SDS-PAGE concentrated gel (amount of one gel made): 0.68ml of ddH was added₂O, 0.17ml of 30% Acrylamide, 0.13ml of 1.0M Tris-HCl (pH6.8), 0.01ml of 10% SDS, 0.01ml of 10% (W/V) ammonium persulfate, 0.001ml of TEMED.

(26) Electrode buffer solution: 1g SDS, 3g Tris, 14.4g Gly were weighed out and finally ddH was used₂O is metered to 1000ml and is prepared for use; alternatively, a 10-fold SDS-free concentrate is prepared, stored at room temperature, and when necessary, 1g of SDS is added to each 1000ml of the dilution, and the resulting mixture is used after being sufficiently dissolved.

Examples

(1) Extraction of protoplasts

Treatment of enzymatic hydrolysate: preparing protoplast enzymolysis solution, slightly shaking to dissolve the solute completely, heating in 55 deg.C water bath for 10min (the water bath needs to be opened in advance for preheating), cooling on ice for 5min, and adding 100 μ l CaCl₂(1M), shaking evenly lightly, and standing temporarily at room temperature for later use;

releasing protoplast: taking 3-3.5g of fresh tender pulp by using a clean forceps, adding the fresh tender pulp into a sterilized culture dish, slightly crushing the pulp, adding the treated cell wall enzymolysis liquid, slightly shaking the pulp uniformly, wrapping the pulp by using tin foil paper to prevent light, placing the pulp on an open-air shaking table at the speed of 70rpm, and carrying out enzymolysis for 2-3 hours at room temperature;

thirdly, separating and collecting protoplast: directly adding equal volume (one part of material is 10ml in a system of 10ml, namely 10ml of W5 buffer solution is added into each part of material) of W5 buffer solution into an upper culture dish for cell wall enzymolysis by a step, gently shaking the material uniformly, filtering the material by using 200-mesh nylon gauze into a 50-ml round-bottom centrifuge tube, washing a filter screen by using 5-10ml of W5 buffer solution, standing the filtered material at room temperature (about 25 ℃) for 20-30 minutes to naturally settle protoplasts, or precipitating the filtered material by using a centrifuge (500rpm, 10min, about 25 ℃) and then carefully sucking supernatant by using 1000 mul of pipette with a smooth shearing gun head, reserving the precipitate, adding 5-10ml of W5 buffer solution into the centrifuge tube for reserving the precipitate, gently shaking the precipitate uniformly, re-suspending the precipitate, standing the precipitate at room temperature (about 25 ℃) for 20-30 minutes to naturally settle the protoplasts, alternatively, the precipitation was carried out by a centrifuge (500rpm, 10min, room temperature, 25 ℃ C.) and the supernatant was carefully aspirated by using 1000. mu.l of a pipette with a smooth shearing tip, and the precipitate was retained. A small amount of protoplasts can be retained in MMG buffer to facilitate observation of the status and quality of the protoplasts.

By detection and calculation, 500-700. mu.l protoplast pellet per 3-3.5g of pulp can be obtained (as shown in FIG. 1).

(2) Extraction of vacuoles

Releasing vacuoles: directly adding 5-6ml of lysis buffer (protoplast obtained from 3-3.5g of pulp is a sample required by extraction vacuole) with heat preservation in 37 deg.C water bath into the protoplast precipitate, shaking, mixing, standing at room temperature for 15 min, and centrifuging;

separating and collecting vacuoles: then 3ml of 4% ficoll solution stored at room temperature was added to each sample, 1ml of vacuole buffer stored on ice was added, the samples were trimmed on a balance and centrifuged (data set: 71000g, 10 ℃, 50 min);

(Care should be taken to avoid mixing the different solution layers when adding liquid)

Observation: after centrifugation, the layers were carefully separated by a pipette with a 100. mu.l smooth, cut tip, according to the separation layer that appeared, transferred to a clean centrifuge tube of appropriate size and marked to avoid mixing, and the solutions from each part were individually pelleted and observed under an optical microscope. The content of vacuoles extracted is, by calculation, about 100/mm². In addition, for easier observation of apple fruit cell protoplasts, 0.1% neutral red (Sigma, cat # N7005) solution (0.1g neutral red, 200. mu.l 1% acetic acid, 50. mu.l chloroform, and finally ddH can be used₂O to 100ml) for 5min, and then the slide was observed under an optical microscope. Observed and calculated, the vacuole extraction amount is about 100 per mm²(as shown in fig. 2).

(3) SDS Polyacrylamide gel electrophoresis

Firstly, processing materials after ultracentrifugation: determining a separation layer containing a large amount of vacuoles by an optical microscope, marking the part, freezing the marked part at-80 ℃, freezing and crushing the vacuoles in the part after a certain time, taking out the part, placing the part on ice to melt, taking 100 mu l of the part into a clean 1.5ml centrifugal tube by using a pipette after the part is completely melted, adding 25 mu l of sample buffer solution into the centrifugal tube, fully and uniformly mixing the buffer solution, clamping the buffer solution in two layers of foam plates, installing an explosion-proof device on a tube opening, boiling the buffer solution in boiling water for 8 minutes, and using the buffer solution after the boiling is finished, wherein if the buffer solution cannot be used immediately, the buffer solution needs to be stored at-20 ℃;

preparing glue: selecting clean and dry filler strip glass and thin glass to be respectively matched into a set, overlapping the two, forming a gap in the middle, vertically placing a glue-making support, aligning the lower ends of the filler strip glass and the thin glass on a flat desktop, then rotationally locking doors at two sides of the glue-making support towards two sides to complete a glue-making unit (note that when fixing glass plates, the overlapped bottoms of the two glass plates are aligned as much as possible, fingers touch the glass plates smoothly, the glass plates are carried out on a flat operating platform as much as possible, otherwise, glue leakage is serious after glue injection, so that subsequent experimental operation is influenced), tightly pressing the bottom of the prepared glue-making unit onto a sealing rubber strip of a glue-making fixing frame, enabling the back edge of the glue-making support to be close to the glue-making fixing frame as much as possible, and opening an upper spring clamp to tightly clamp the upper edge of the glass plates (if the clamp is not tight, or the performance of the rubber strip is poor, so that subsequent glue leakage is caused), and then the glass plate is fixed. Preparing lower layer separation gel solution according to the formula, injecting the lower layer separation gel solution into a gap between two glass plates by using a 1000 mu l pipette, and using ddH₂And O, filling the rest space and driving out bubbles generated during glue injection. Waiting for the separation glue to solidify (after about 20-30 minutes, an obvious boundary line can appear between the water of the lower layer of separation glue and the water of the upper layer, observing the leakage degree of the glue, determining to continue subsequent work or re-manufacture according to the height of the boundary line, checking a fixing device to replace accessories which are possibly influenced if the re-manufacture is needed), pouring the water of the upper layer of separation glue if the lower layer of separation glue is available, carefully sucking the water which is not easy to pour out in the lower layer of separation glue by using filter paper, and taking care not to poke the separation glue; immediately after the top water treatment, the top concentrated gel was formulated and injected into the gap between the two glass plates using a 1000. mu.l pipette (care was taken to keep bubbles as small as possible, if any, they were aspirated by the pipette or expelled by a syringe) immediatelyInserting a comb with required thickness and tooth number, and using after the comb is solidified;

preparing an electrode buffer solution: 100ml of 10-fold electrode buffer stored at room temperature was diluted to 1000ml, and 1g of SDS powder was added thereto and dissolved by stirring sufficiently for further use. (SDS powder is easily dusted and harmful to inhalation, the powder should be applied by wearing a mask and quickly manipulated to avoid inhalation);

assembling the electrophoresis device: after the glue is solidified, taking out the glue making unit without pulling out the comb, opening a contraction door on the glue making bracket, taking out the glass plate from the glue making bracket, and moving the glass plate into an electrophoresis tank; respectively loading a group of finished glass plates (a thin glass plate facing to the inner side) and a substitute plastic sheet into two sides of an electrode frame, placing the bottoms of the glass plates and the substitute plastic sheet on a support of the electrode frame, pinching and clamping to form a complete sealing groove (when the glass plates and the plastic sheet are fixed, small grooves at the left upper corner and the right upper corner of the glass plates and the plastic sheet are aligned with a bulge on a fixing device so as to prevent leakage after an electrode buffer solution is added); inserting the assembled electrode frame into the fixing frame in the vertical groove, and confirming that the electrode frame corresponds to the positive and negative electrodes of the vertical groove and the clamping groove;

glue running: filling an electrode buffer solution in the inner tank, overflowing a small part of the buffer solution to the outer tank, carefully pulling out the comb, adding the protein sample and the protein marker into the comb well according to a certain sequence, wherein each hole is 10 mu l, and the concentration of the protein sample can be properly adjusted; finally, the upper cover is covered, the anode and the cathode of the electrode frame are ensured to be corresponding to the anode and the cathode of the groove cover all the time, the electrophoresis apparatus is switched on and the voltage is adjusted (the voltage is adjusted to be 110V before the sample runs to the interface of the concentrated gel and the separation gel, the voltage is adjusted to be 130V when the sample reaches the interface, and the electrophoresis apparatus is stopped until the sample is observed to be close to the bottom of the separation gel);

sixthly, dyeing, observing and decoloring: after the application, the power is cut off, the electrophoresis tank and the electrode holder are washed by clear water, the electrophoresis tank and the electrode holder are laid on a table for airing, a thin glass plate is carefully pried open by a glue shovel, the albumin glue is carefully taken out, the albumin glue is placed in a large-sized culture dish, an appropriate amount of SDS-PAGE albumin glue dye solution (purchased from Beijing Rui Borxing science and Biotechnology Co., Ltd., cat: RB010-500, lot. No: RBA00117) which can not pass the albumin glue is poured in, at least 5 minutes of dyeing is carried out, whether a dyed protein strip appears or not is observed under an instrument, if a remarkable strip appears, the glue is placed in a decoloring solution for decoloring for at least 2 hours so as to facilitate observation (as shown in figure 3). The vacuolar protein extraction concentration is approximately 1. mu.g/ml.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.