Method and device for non-destructively recovering protein from polyacrylamide gel
By immobilizing polyacrylamide gel strips loaded with target proteins on agarose gel and electrophoresis under low temperature and low pressure conditions, combined with the device design of a container and a pull-out sealing structure, the problems of expensive equipment, complicated operation, low efficiency and high risk of cross-contamination of existing protein recovery devices are solved, and efficient and non-destructive protein recovery and sequencing are achieved.
Patent Information
- Application Number
- CN202510921196.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-04
- Publication Date
- 2025-10-21
AI Technical Summary
Existing protein recovery devices suffer from problems such as high equipment cost, complex operation, low efficiency, difficulty in temperature control, high risk of cross-contamination, and low efficiency of protein electrophoretic migration.
Agarose gel was used to immobilize polyacrylamide gel strips loaded with target proteins. Electrophoresis was performed under low temperature and low pressure conditions. Combined with the device design of a container and a pull-out sealing structure, the non-destructive release and recovery of the target proteins were achieved.
It achieves efficient and non-destructive protein recovery, precise temperature control, reduces the risk of cross-contamination, improves electrophoretic migration efficiency, and supports efficient recovery and sequencing of multiple samples.
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Figure CN120818006A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to a method and device for lossless protein recovery from polyacrylamide gel, belonging to the technical field of protein separation by electrophoresis. Background Art
[0002] During protein sample purification, after the protein stock solution is concentrated, polyacrylamide gel electrophoresis is used to separate proteins of varying molecular weight. Specific proteins often aggregate in the polyacrylamide gel, forming distinct protein bands. Conventional methods for recovering protein bands from polyacrylamide gels involve gel dissolution or extraction, resulting in recoveries generally below 40%. These methods are also costly, involve corrosive and toxic reagents, and generate significant environmental impacts from waste. For example, a macroporous resin can be combined with peanut hydrolyzed protein peptides to remove impurities such as sugars, thereby concentrating the peanut hydrolyzed protein peptides and removing impurities. Alternatively, multiple tubes can be run in a single electrophoresis bath, but this poses the risk of cross-contamination between samples. Simply placing polyacrylamide gel blocks on a sieve plate can cause block displacement during electrophoresis, especially when the magnetic stirrer at the bottom of the device is running. This can affect protein electrophoretic migration efficiency and increase the risk of cross-contamination. Furthermore, temperature control is difficult due to the use of a single electrophoresis tank, and poor temperature control can easily lead to protein degradation.
[0003] Therefore, current protein recovery devices often have the following problems:
[0004] 1) The equipment is relatively expensive and not suitable for experimental promotion;
[0005] 2) The dialysis membrane needs to be installed, and the connection between the dialysis bag and the tube increases the complexity;
[0006] 3) Only one sample can be operated at a time, which is inefficient;
[0007] 4) Temperature control is difficult and the recovery efficiency is low;
[0008] 5) Polyacrylamide gel blocks are randomly distributed in the device and cannot be fixed, resulting in low protein electrophoretic migration efficiency. Summary of the Invention
[0009] In view of the above technical drawbacks of protein recovery, the present application first provides a method for lossless recovery of proteins from polyacrylamide gel, which can not only achieve the fixation and direction adjustment of polyacrylamide gel, but also has high efficiency of protein electrophoretic migration; the recovery process is physical (electric field) release, and also has the nature of lossless protein recovery.
[0010] Specifically, this application is implemented through the following solutions:
[0011] The invention discloses a method for losslessly recovering proteins from polyacrylamide gel. The method comprises fixing a polyacrylamide gel strip loaded with a target protein with agarose gel, and performing electrophoresis under low temperature and low pressure conditions of 1-5°C and 0.5-1.5V / cm. The target protein can be losslessly released from the polyacrylamide gel into the electrophoresis solution, thereby completing the physical recovery of the target protein from the polyacrylamide gel. The concentration of the agarose gel is 0.3-0.8%.
[0012] Furthermore, as a preference:
[0013] The low temperature and low pressure conditions refer to 4° C., 1.0 V / cm, and an agarose gel concentration of 0.6%.
[0014] The electrophoresis fluid is a non-denaturing polyacrylamide gel electrophoresis fluid.
[0015] The target protein is peanut SOD protein. In this case, the polyacrylamide gel band blocked by the target protein is obtained by staining peanut protein for superoxide dismutase (SOD) activity and cutting the gel.
[0016] The above scheme uses electrophoresis under low temperature and low pressure conditions to release the target protein loaded on the polyacrylamide gel strip physically (electric field). The solution can then be concentrated for direct protein sequencing. The above recovery method can also achieve precise temperature control, which has good practicality and accuracy.
[0017] The applicant also provides a device for losslessly recovering proteins from polyacrylamide gel, comprising a container, wherein the two ends of the container are respectively connected to a positive electrode and a negative electrode, a gel block blocking net and a plug-in sealing structure are provided in the container, the plug-in sealing structure is located on one side of the negative electrode, and one end of the plug-in sealing structure is connected to the negative electrode, and the other end is provided with an opening, the gel block blocking net is located at the opening, and the plug-in sealing structure is filled with agarose gel for fixing the polyacrylamide gel strips; the electrophoresis liquid containing the polyacrylamide gel strips loaded with the target protein is injected into the container from the positive electrode side and enters the plug-in sealing structure through the opening.
[0018] Preferred:
[0019] The positive electrode and the negative electrode are connected to a DC power supply or an AC power supply.
[0020] A plurality of the containers are connected in parallel to form a matrix-type lossless protein recovery device.
[0021] The container is connected to a proteome sequencing device.
[0022] The above-mentioned device can be either single and portable (i.e., in the case of a single container) or integrated in a parallel matrix. In particular, the latter can be connected in series with the proteome sequencing facility, which not only realizes the separate recovery of multiple samples, but also realizes the precision and massive operation of the recovery process. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.
[0024] Figure 1 This is a schematic diagram of the structure of the non-destructive protein recovery device of this application.
[0025] Reference numerals in the figure: 1. container, 2. gel block blocking net, 3. plug-in sealing structure, A: homogeneous electrophoresis solution containing polyacrylamide gel strips loaded with target protein;
[0026] Figure 2 This is the SOD activity staining pattern of non-denaturing polyacrylamide gel of peanuts from different sources.
[0027] M: protein marker, A: peanuts treated with multi-enzyme solution (Xinxiang, Henan), B: peanuts not treated with multi-enzyme solution (Xinxiang, Henan), C: white peanuts (DuShang Supermarket, Xiasha, Hangzhou) and multi-enzyme solution-treated peanuts (Xinxiang, Henan), D: white peanuts (Wumi, Jinsha Lake, Xiasha, Hangzhou), E: red peanuts (Wumi, Jinsha Lake, Xiasha, Hangzhou), F: white peanuts (2024 seasonal peanuts, Xincai, Henan), 1 to 5 on the right indicate five SOD protein staining bands;
[0028] Figure 3 This is the electrophoresis staining diagram of peanut SOD protein activity (NBT staining method);
[0029] Figure 4 This is a diagram of the electrophoresis device for SOD protein band excision and recovery.
[0030] A: Agarose gel containing polyacrylamide gel strips, B1 is strip 1, B2 is strip 2 (multiple strips mixed),
[0031] B: Schematic diagram of electrophoresis facilities in the electrophoresis tank;
[0032] Figure 5 This is the detection result of protein recovery from SOD activity staining bands in polyacrylamide gel non-denaturing electrophoresis.
[0033] (A) Laboratory testing, (B) Polyacrylamide gel denaturing electrophoresis testing by the sequencing company before protein sequencing,
[0034] CK is the peanut extract, B1 and B2 are the proteins recovered from the gel excision of the polyacrylamide gel electrophoresis protein band;
[0035] Figure 6 is the abundance (expression level) of six SOD-related proteins in three samples,
[0036] B1 and B2 are the recovered proteins from the gel excision of the electrophoretic protein band, CK is the peanut extract, CCS: SOD copper molecular chaperone;
[0037] Figure 7 The transcriptional profiles of 5 SOD-related genes in peanut seed SOD proteome sequencing along with different developmental stages and organs of peanuts were analyzed.
[0038] TPM: number of target sequences per million transcript reads, CCS: SOD copper chaperone. DETAILED DESCRIPTION
[0039] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the technical solutions in the embodiments of this application will be further described in detail below in conjunction with the drawings in the embodiments of this application. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit the technical solutions of this application. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of this application.
[0040] This example uses peanut seed SOD protein as the target protein to illustrate the present application scheme.
[0041] During the recycling process, the device structure corresponding to this application is as follows Figure 1As shown: it includes a container 1, the two ends of the container 1 are respectively connected to the positive electrode and the negative electrode, a gel block blocking net 2 and a plug-in sealing structure 3 are arranged in the container 1, the plug-in sealing structure 3 is made of insulating material, and is located on the side of the container 1 close to the negative electrode, one end of the plug-in sealing structure 3 is connected to the negative electrode, and the other end is provided with an opening recessed toward the negative electrode side, the opening is covered with the gel block blocking net 2, and the plug-in sealing structure 3 is filled with agarose gel; the homogeneous electrophoresis liquid A containing the polyacrylamide gel strip loaded with the target protein is injected into the container 1 from the positive electrode side and enters the plug-in sealing structure 3 through the opening. During the recovery process, the homogeneous electrophoresis liquid A containing the polyacrylamide gel strip loaded with the target protein permeates from the positive electrode side to the negative electrode side. After the large gel block is blocked on the right side by the gel block blocking net 2, the remaining electrolyte enters the plug-in sealing structure 3. The agarose gel fixes the polyacrylamide gel block, and the recovered target protein returns to the right side, forming an area on the left side (dark part) for fixing the polyacrylamide gel block, and the right side (colorless part) for collecting the target protein fragments.
[0042] The device can be divided into three parts: (1) The electrode part includes a positive electrode and a negative electrode, which are connected to an external power source. It can be designed to be an AC power source or a DC power source according to the specific scenario. The DC power source can be directly driven by a battery, which improves its portability; (2) The plug-in sealing structure 3 (insulating material): The plug-in sealing structure 3 is connected to the electrode (negative electrode) at one end and is provided with an opening at the other end. When the plug-in sealing structure 3 is inserted into the container 1, its edge is sealed and does not leak liquid. At the same time, it is also convenient to fix the polyacrylamide gel block with agarose glue. In this embodiment, the concentration of agarose glue is 0.6%, the polyacrylamide gel block is perpendicular to the inner wall of the container 2, and the total length of the container 1 is 0.6 to 1.0 cm. (3) Protein fragment collection part: This part can be a part of the container 1, or it can be a mechanism that can be installed in the container 1 in a pluggable manner like the pluggable sealing structure 3, but its material is consistent with the pluggable sealing structure 3, one end is connected to the electrode (positive electrode), and the other end is open. The open end forms a chimeric sealing structure with the opening of the pluggable sealing structure 3. The interior of the protein fragment collection part is mainly electrophoresis fluid.
[0043] The operating environment of the above device is low temperature (4°C) and low voltage (1.0V / cm), which can not only separate proteins without loss, but also form a matrix by connecting multiple containers 1 in parallel. When connected to the proteome sequencing equipment, the recovered proteome (colorless part) can also be sequenced in segments on a large scale.
[0044] The recycling method is described in detail below.
[0045] Before recycling, we analyzed the SOD activity staining patterns of peanuts from different sources on non-denaturing polyacrylamide gels. Figure 2As shown. The SOD type of band 1 is unknown and may be a protein complex; band 2 and the three bands below it (about four bands in total) were identified as Cu / Zn-SOD. In order to accurately identify the types of these two types of SOD proteins, we used a proteomic sequencing strategy to determine the type of peanut SOD protein. The specific steps include non-denaturing electrophoresis, SOD activity staining, gel cutting, agarose electrophoresis protein extraction, vacuum rotary evaporation concentration, identification, proteomic sequencing, and result analysis. Finally, we obtained six protein sequences, one of which was Mn-SOD, four were Cu / Zn-SOD, and one was a SOD copper chaperone (CCS).
[0046] 1) Peanut protein polyacrylamide gel electrophoresis and SOD activity staining
[0047] The newly extracted peanut SOD protein crude extract was subjected to non-denaturing polyacrylamide gel electrophoresis and SOD enzyme activity staining. The results are as follows Figure 3 As shown, Figure 2 The experimental results were consistent, B1 was a complex that may contain SOD protein; B2 contained Cu / Zn-SOD protein (since the four protein bands here were identified as Cu / Zn-SOD, they were collected together and collectively referred to as B2).
[0048] 2) Peanut SOD activity staining band cutting and SOD protein recovery
[0049] Cut B1 and B2 separately (do not crush the strips), and divide the intact strips into sections according to the inner edge of the smallest gel plate for agarose gel electrophoresis. Then use 10x non-denaturing polyacrylamide gel electrophoresis buffer (Tris: 60.6g, methyl sulfonate (MES): 97.6g, ethylenediaminetetraacetic acid: 3g, add deionized water to 1 liter, and dilute 10 times before use) to prepare agarose gel (0.6%). When the gel solution is about to solidify (about 60℃), carefully insert the polyacrylamide gel strip vertically into the gel. For details, see Figure 4 (A) After solidification, excess agarose was removed along the inserted strip.
[0050] After 30 minutes, take out the solidified agarose gel from the gel plate, remove the excess gel from the gel plate, and leave only the gel where the polyacrylamide gel strip is located. Then put the gel plate with the gel strip into a 3.5KDa pre-treated dialysis bag with a diameter of 77mm, seal one end with a clamp, and add non-denaturing polyacrylamide gel electrophoresis fluid to the other end, just enough to cover the gel surface (2mm above the gel surface), and seal the other end with a clamp. After sealing, put it into the horizontal electrophoresis tank (the polyacrylamide gel strip end is at the negative pole), and add non-denaturing polyacrylamide gel electrophoresis fluid to 2mm above the gel in the gel tank. For details, see Figure 4(B) in the electrophoresis tank. Then, place the electrophoresis tank in a 4°C environment. Once the temperature is constant, connect the electrophoresis instrument and adjust the voltage to 30V (the distance between the cathode and anode electrodes in the electrophoresis tank is 30cm, resulting in a reaction voltage of 30V / 30cm = 1.0V / cm). Run the electrophoresis for 12 hours. After the electrophoresis is complete, collect the electrophoresis solution containing the target protein from the dialysis bag.
[0051] 3) Peanut SOD protein concentration and polyacrylamide gel electrophoresis identification
[0052] Use a vacuum centrifugal concentrator to concentrate the electrophoresis solution, divide the electrophoresis solution into 2ml centrifuge tubes, open the lid and place them in a vacuum rotary evaporator, centrifuge for 40 minutes, and concentrate the 4ml solution to 0.2ml. Use denaturing polyacrylamide gel electrophoresis to analyze the concentrated protein. Figure 5 (A): Clear bands are evident in both B1 and B2, demonstrating the feasibility of the gel electrophoresis and subsequent recovery method. Furthermore, B2 exhibits significantly fewer bands, with two main bands, indicating that the Cu / Zn-SOD protein is a multi-subunit protein and may also contain heterogeneous Cu / Zn-SOD proteins forming a complex, along with other proteins. B1 exhibits more bands with a higher molecular weight, indicating that it contains multiple proteins, including SOD. Protein quality testing at a protein sequencing company was consistent with this result. Figure 5 These results indicate that the method of this embodiment can effectively recover proteins (while the protein recovery kit using a polyacrylamide gel cannot achieve this effect). At the same time, this result also indirectly confirms that the protein recovery device developed in this application can be used for the recovery of other proteins.
[0053] 4) Proteome sequencing and analysis
[0054] Protein sequencing was performed by the Protein Sequencing Department of Hangzhou Lianchuan Biotechnology Co., Ltd. using data-independent acquisition (DIA) scanning mode (https: / / www.nature.com / articles / s41592-019-0638-x, version 1.9.1).
[0055] The sequenced polypeptide chains were aligned with the Uniprot peanut (Arachis hypogaea) proteome data (downloaded on December 4, 2024), and a total of 6 SOD-related proteins were obtained, including five SOD proteins and one copper ion SOD molecular chaperone protein CCS (see Tables 1 and 2). Only five SOD proteins are considered here. Among the five SOD proteins, only one is a Mn / Fe-SOD protein, and the other four are Cu / Zn-SOD proteins ( Figure 2The rightmost side of the figure shows five active bands, corresponding to these five proteins).
[0056] Table 1: Annotation information of SOD-related proteins in peanut seeds
[0057]
[0058]
[0059] Table 2: Information of six peanut SOD-related proteins
[0060]
[0061] The paper "Identification and analysis of SOD family genes in peanut (Arachis hypogaea L.) and their potential roles in stress responses" systematically analyzed the peanut protein family genes and obtained detailed information on 25 peanut SOD genes and proteins. According to the protein sequencing results, the six SOD-related proteins obtained had a high degree of sequence identity with six of the 25 genes. Therefore, we named the six peanut SOD-related genes (see Table 2) and further analyzed the abundance of these six SOD-related proteins in three samples, as shown in the following figure. Figure 6As shown, in general, the abundance of the six sample proteins is lowest in CK (except A0A45B5S3 (Mn / Fe-SOD), which is higher in CK (54,400) than in B2 (39,800), and 535,000 in B1), indicating that these proteins are enriched to a certain extent in B1 and B2. From the B1 and B2 samples, the abundance of protein fragments of these six SOD proteins all appeared, indicating that the SOD in the two samples may be a heterologous complex or a homologous complex. Obviously, Q1HDS7 (Cu / Zn-SOD) and A0A445CNQ2 (Cu / Zn-SOD) are mainly enriched in B2, at 34.1828 million and 34.0904 million, respectively. At the same time, the molecular weight of the two proteins is not much different, both are 152aa, and the amino acid motif homology is very high. Through comparison, only 3 amino acids are different. This indicates that these two SOD proteins are abundant in peanut seeds and play a crucial role in protecting seed viability. A0A445E4U2 (Cu / Zn-SOD), A0A445B1R6 (Cu / Zn-SOD), and A0A445ER89 (CCS, a copper chaperone for SOD) were most highly enriched in B2, in that order. A0A445B1R6 (Cu / Zn-SOD) was particularly enriched in B1 and B2 (90 kDa), with a peak concentration ranging from 512 to 49,700 in B1 and 65,600 in B2, compared to CK. A0A45B5S3 (Mn / Fe-SOD) was primarily enriched in B1 (approximately 200 kDa), reaching 534,600 kDa, indicating that the predominant SOD species in the B1 band is Mn / Fe-SOD. These findings suggest that SODs likely function by forming multimers on their own or by binding to other proteins. Four SODs, including Q1HDS7 (Cu / Zn-SOD), A0A445CNQ2 (Cu / Zn-SOD), A0A445E4U2 (Cu / Zn-SOD), and A0A445B1R6 (Cu / Zn-SOD), showed high enrichment folds, suggesting their heat resistance. Furthermore, the abundance of Mn / Fe-SOD and Cu / Zn-SOD types was over 100 times greater, indicating that Cu / Zn-SODs predominate in peanut seeds. Although A0A445ER89 (CCS, a copper chaperone for SODs) was most highly enriched in B2, the difference was not significant compared to B1 and CK, indicating that CCS is present in both B1 and B2 bands. It is likely that CCS acts as a universal copper-binding protein, assisting Cu / Zn-SOD in its function.
[0062] Yu et al. (2023) analyzed the SOD gene family in peanut (Arachis hypogaea L.) at the genomic level and identified 25 SOD genes, including 16 Cu / Zn-SODs, 6 Fe-SODs, and 3 Mn-SODs. We compared the six SOD-related protein sequences obtained by protein sequencing with the 25 sequences from Yu et al. (2023) and found that Q1HDS7, A0A445E4U2, A0A445ER89, and A0A445B5S3 were identical to AhSOD15-Cu, AhSOD03-Cu, AhSOD02-Cu, and AhSOD09-Mn, respectively. A0A445CNQ2 differed from AhSOD06-Cu at the carbon terminus, and A0A445B1R6 had an additional nitrogen-terminal sequence compared to AhSOD10-Cu. Q1HDS7 and A0A445CNQ2 are gene duplications, and their promoter regions contain numerous drought-inducibility elements (MBSs) and stress-response elements (STREs). The A0A445B1R6 promoter contains four ABRE domains, and A0A445E4U2 contains five ARE domains and five CGTCA motifs (Yu et al. 2023). These findings further demonstrate the complexity and diversity of the peanut SOD family genes, reflecting the complexity of their gene expression regulation.
[0063] The document "The genome sequence of segmental allotetraploid peanut (Bertioli DJ, Jenkins J, Clevenger J, Dudchenko O, Gao D, Seijo G, Leal-Bertioli SCM, Ren L, Farmer AD, Pandey MK et al: Arachis hypogaea. Nature Genetics 2019, 51(5): 877-884.)" describes the peanut genome sequencing and related databases. The protein recovered in this application was compared with the SOD protein sequence, and the transcription profile information of these genes was obtained based on the data, such as Figure 7 As shown, the results show that the recovery method of the present application can obtain peanut SOD protein with detectable sequence information.
[0064] Six proteins (Table 2) were retrieved from the Uniprot database (https: / / www.uniprot.org / ) and linked to the peanut database (https: / / www.peanutbase.org / ) (Bertioli et al. 2019). The peanut database was then linked to the gene-specific genomic and transcriptomic data (https: / / mines.legumeinfo.org / arachismine / begin.do) to obtain the developmental transcriptome expression profiles of five of these genes ( Figure 7 , because the sampling points of the transcriptome map data have 3, 2, and 1 biological replicates, the figure only shows the average value of TPM). Another gene (A0A445B1R6 (Cu / Zn-SOD)) did not find matching data in the peanut genome annotation data, but there are records of this gene in UniProt and NCBI. There may be certain errors in the annotation of this sequence, which requires further analysis. From these 5 genes: the expression of the SOD copper molecular chaperone gene (A0A445ER89) is at a low level at all stages of peanut development, which is likely to be its background expression. However, the protein content in the seeds is relatively high ( Figure 6 ), it is very likely that there is no positive correlation between the transcription level of this gene and the protein content. It is very likely that this protein has a certain stability in the cell or is protected and modified. Further research is needed to solve this mystery. At each stage of seed development, the expression of Cu / Zn-SOD (Q1HDS7 and A0A445CNQ2) genes was significantly upregulated (reaching the highest value at Seed Pattee stage 8), and slightly downregulated at the later stage (Seed Pattee stage 10). The expression levels in other stages of peanut growth and development or tissues and organs were relatively low, indicating that the genes of Q1HDS7 and A0A445CNQ2 are mainly expressed in seeds, which is consistent with the protein content in seeds ( Figure 6 ) are highly consistent. At the same time, the amino acid sequences of the two gene proteins are highly consistent, with only 3 amino acid differences, indicating that the two genes may have undergone gene duplication events during evolution, which did not affect the expression patterns of the two genes (Yu et al. 2023). The expression profile of the Mn / Fe-SOD (A0A445B5S3) gene varies greatly. The highest expression level in seeds appears in Seed Pattee stage 6, and it has higher expression in Pod Pattee stage 3 / 4, Nodules, and Gyn tip Pattee stage 1. However, it is lower than the highest expression of Q1HDS7 and A0A445CNQ2 genes in seeds, which is also related to its protein amount ( Figure 6). Protein sequence subcellular localization prediction indicates that Mn / Fe-SOD is a mitochondrial protein, and its expression fluctuation is likely related to peanut development and the activity of cells in various tissues and organs. A0A445E4U2 (Cu / Zn-SOD) is expressed at trace levels in seeds but is primarily expressed in leaves, suggesting that its function may be related to biochemical processes such as free radical scavenging in leaves.
[0065] In summary, when using a pre-ordered chemical kit (polyacrylamide gel recovery kit) to recover peanut SOD protein, we observed low recovery efficiency and degradation. However, the device and method of the present application can recover protein from polyacrylamide gels with high quality (high recovery rate, no degradation). The peanut SOD protein recovery process described above demonstrates that the target protein recovered by the present invention is of sufficient quality to be directly sequenced. Proteomic sequencing of the recovered protein revealed five SOD protein sequences and one SOD protein copper ion binding chaperone sequence; the former includes one Mn-SOD type and four Cu / Zn-SOD type proteins.
[0066] The above-described embodiments merely represent several feasible implementation methods of the present invention. The description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention. The embodiments are not intended to limit the scope of protection in the claims of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention. Any equivalent implementation or modification that does not depart from the scope of the present invention should be included in the technology of the present invention.
Claims
1. A method for lossless protein recovery from polyacrylamide gel, characterized in that: The target protein is fixed on a polyacrylamide gel strip with agarose gel, and electrophoresis is performed under low temperature and low pressure conditions of 1-5°C and 0.5-1.5V / cm, so that the target protein can be released from the polyacrylamide gel into the electrophoresis solution without loss, thereby completing the physical recovery of the target protein in the polyacrylamide gel. The concentration of the agarose gel is 0.3-0.8%.
2. The method for lossless protein recovery from polyacrylamide gel according to claim 1, wherein: Low temperature and low pressure conditions refer to 4°C, 1.0 V / cm, and an agarose gel concentration of 0.6%.
3. The method for lossless protein recovery from polyacrylamide gel according to claim 1, wherein: The electrophoresis fluid is a non-denaturing polyacrylamide gel electrophoresis fluid.
4. The method for lossless protein recovery from polyacrylamide gel according to claim 1, wherein: The target protein is peanut SOD protein.
5. The method for lossless protein recovery from polyacrylamide gel according to claim 4, characterized in that: The polyacrylamide gel strips are obtained by staining peanut protein with SOD enzyme activity and cutting the gel.
6. A device for lossless protein recovery from polyacrylamide gel, characterized by: The invention comprises a container, wherein the two ends of the container are respectively connected to the positive electrode and the negative electrode; a gel block blocking net and a plug-in sealing structure are arranged in the container; the plug-in sealing structure is located on one side of the negative electrode, and one end of the plug-in sealing structure is connected to the negative electrode, and the other end is provided with an opening; the gel block blocking net is located at the opening; the plug-in sealing structure is filled with agarose gel for fixing polyacrylamide gel strips; the electrophoresis liquid containing the polyacrylamide gel strips loaded with the target protein is injected into the container from the positive electrode side and enters the plug-in sealing structure through the opening.
7. The device for lossless protein recovery from polyacrylamide gel according to claim 6, characterized in that: The positive electrode and the negative electrode are connected to a DC power supply or an AC power supply.
8. The device for lossless protein recovery from polyacrylamide gel according to claim 6, characterized in that: A plurality of the containers are connected in parallel to form a matrix-type lossless protein recovery device.
9. The device for lossless protein recovery from polyacrylamide gel according to claim 6, characterized in that: The container is connected to a proteome sequencing device.