Animal cell membrane protein and cytoplasm protein separation and extraction reagent and application

Abstract

The application discloses an animal cell membrane protein and cytoplasm protein separation and extraction reagent and application, and belongs to the technical field of protein separation and extraction. The membrane protein and cytoplasm protein separation and extraction reagent provided by the application can realize separation and extraction of animal cell membrane protein and cytoplasm protein without using complex methods such as repeated freezing and thawing of liquid nitrogen to lyse cells, is simple to operate, efficient and convenient, has high extraction efficiency on membrane protein, and can effectively reduce cross contamination of cytoplasm protein in cell membrane components.

Description

Technical Field

[0001] This invention belongs to the field of protein separation and extraction technology, specifically relating to a reagent for the separation and extraction of animal cell membrane proteins and cytoplasmic proteins and its application. Background Technology

[0002] Biological membranes are crucial barriers separating cells from their external environment and are also vital sites for various intracellular physiological processes. Membrane proteins, as core components of biological membranes, not only participate in basic physiological processes such as substance transport, signal transduction, and energy conversion, but also play important roles in complex life activities such as cell recognition, immune responses, and cell differentiation. Ion channels, G protein-coupled receptors (GPCRs), and transporters, among other types of membrane proteins, hold critical positions in cellular physiological activities and have become highly promising drug targets. Therefore, research on membrane proteins has significant theoretical and practical value, especially in the field of new drug development.

[0003] Functional studies of membrane proteins require the enrichment of structurally intact membrane proteins from the cell membrane or endometrial system. Membrane proteins are anchored to the membrane structure by numerous hydrophobic amino acid residues, making them difficult to dissolve in aqueous buffer systems, and exhibiting characteristics such as low abundance and strong hydrophobicity. Given the strong hydrophobicity of membrane proteins, removing or reducing the cytoplasmic protein component from total cellular protein requires the use of detergents or commercially available kits for membrane protein separation and purification. Common commercially available membrane protein extraction kits typically face the following three problems: First, the operation steps are complex, time-consuming, and labor-intensive (e.g., using homogenizers or repeated freeze-thaw cycles in liquid nitrogen to lyse cells, leading to high subjectivity in experimental results); second, the membrane / cytoplasm separation effect is poor, resulting in significant contamination of the membrane protein component with cytoplasmic proteins; and third, the extraction efficiency of membrane proteins is low, resulting in a low total amount of the target membrane protein.

[0004] To address the aforementioned problems, this invention provides a reagent-based separation and extraction reagent for membrane and cytoplasmic proteins. This reagent enables the separation and extraction of membrane and cytoplasmic proteins without requiring physical cell treatment, offering high efficiency and convenience. The reagent provided by this invention not only achieves high extraction efficiency for membrane proteins but also effectively reduces cross-contamination of cytoplasmic proteins within cell membrane components. Summary of the Invention

[0005] The present invention aims to provide a reagent for separating and extracting membrane proteins and cytoplasmic proteins in animal cells. This reagent can efficiently separate and extract membrane proteins and cytoplasmic proteins, and effectively reduce cross-contamination between membrane and cytoplasmic proteins. Another objective of the present invention is to provide a kit for separating and extracting membrane proteins and cytoplasmic proteins in animal cells. In addition to the reagents used for extraction and separation, the kit also includes a device for storing the reagents. A further objective of the present invention is to provide a method for separating and extracting membrane proteins and cytoplasmic proteins using the reagents.

[0006] In a first aspect, the present invention provides a reagent for separating and extracting membrane proteins and cytoplasmic proteins, characterized in that the reagent is composed of extraction reagent A, extraction reagent B and extraction reagent C.

[0007] Extraction reagent A includes Tris, NaCl, and EDTA; Extraction reagent B includes Tris, NaCl, MβCD, Digitonion, Aprotinin, Leupeptin, and Pepstatin.

[0008] Extraction reagent C includes Tris, NaCl, LMNG, GDN, SCS, PI(4,5)P2, DNase I, Aprotinin, Leupeptin, and Pepstatin.

[0009] Preferably, the extraction reagent A consists of the following components at final concentrations: 10-20 mM Tris, 100-200 mM NaCl, and 0.5-1.5 mM EDTA.

[0010] Extraction reagent B consists of the following components at final concentrations: 10-20 mM Tris, 100-200 mM NaCl, 0.05%-0.15% (w / v) MβCD, 0.05%-0.15% (w / v) Digitonion, 0.5-1 μg / mL Aprotinin, 1.5-2.5 μg / mL Leupeptin, and 1.5-2.5 μg / mL Pepstatin.

[0011] Extraction reagent C consists of the following components at final concentrations: 10-20 mM Tris, 100-200 mM NaCl, 0.75%-1.25% (w / v) LMNG, 0.25%-0.35% (w / v) GDN, 0.5-1.5 mM SCS, 50-100 μM PI(4,5)P2, 1.5-2 μg / mL DNase I, 0.5-1 μg / mL Aprotinin, 1.5-2.5 μg / mL Leupeptin, and 1.5-2.5 μg / mL Pepstatin.

[0012] In one embodiment of the invention, extraction reagent A consists of the following components at final concentrations: 20 mM Tris, 150 mM NaCl, and 1 mM EDTA.

[0013] In one embodiment of the invention, the extraction reagent B consists of the following components at final concentrations: 20 mM Tris, 150 mM NaCl, 0.15% (w / v) MβCD, 0.05% (w / v) Digitonion, 1 μg / mL Aprotinin, 2 μg / mL Leupeptin, and 2 μg / mL Pepstatin.

[0014] In one embodiment of the present invention, the extraction reagent C consists of the following components at final concentrations: 20 mM Tris, 150 mM NaCl, 1.25% (w / v) LMNG, 0.25% (w / v) GDN, 1 mM SCS, 50 μM PI(4,5)P2, 2 μg / mL DNase I, 1 μg / mL Aprotinin, 2 μg / mL Leupeptin, and 2 μg / mL Pepstatin.

[0015] Furthermore, the pH value of Tris and / or EDTA used in preparing extraction reagent A is 7.5-8.0, the pH value of Tris used in preparing extraction reagent B is 7.5-8.0, and the pH value of Tris used in preparing extraction reagent C is 7.5-8.0; preferably, the pH value of Tris and / or EDTA used in preparing extraction reagent A is 8.0, the pH value of Tris used in preparing extraction reagent B is 7.5, and the pH value of Tris used in preparing extraction reagent C is 7.5.

[0016] Furthermore, the aprotinin, leupeptin, and pepstatin used in the preparation of extraction reagent B were all added before use; the DNase I, aprotinin, leupeptin, and pepstatin used in the preparation of extraction reagent C were all added before use.

[0017] The English and Chinese names and functional explanations of the components involved in the extraction reagent provided by this invention are as follows: EDTA: An excellent metal ion chelating agent that can effectively remove Ca from solution. 2+ Mg 2+ Fe 2+ Fe 3+ Metal ions, etc.

[0018] MβCD: Methyl-β-cyclodextrin, can disrupt the lipid raft structure on the cell membrane surface, thereby altering the fluidity and permeability of the cell membrane.

[0019] Digitonion: A natural detergent that increases cell membrane permeability by binding to cholesterol molecules.

[0020] Aprotinin is a serine protease (BPTI) inhibitor isolated from bovine lung. It inhibits chymotrypsin, trypsin, kallikrein, and plasmin.

[0021] Leupeptin is a broad-spectrum protease inhibitor with cell membrane permeability that effectively inhibits serine, cysteine, and threonine proteases.

[0022] Pepstatin is a specific, orally active aspartic protease inhibitor produced by actinomycetes.

[0023] LMNG: Lauryl Maltose Neopentyl Glyco, is a nonionic detergent commonly used in biochemical research. It effectively dissolves and stabilizes membrane proteins, exhibiting excellent performance in preserving their native structure, and is widely used in the purification and structural studies of membrane proteins.

[0024] GDN: Glyco-diosgenin, is a synthetic surfactant and detergent used to extract proteins from membranes for structural and functional studies, as well as for single-particle cryo-electron microscopy studies of membrane proteins.

[0025] SCS: Sodium cholesteryl sulfate, a component that mimics the structure of the cell membrane surface.

[0026] PI(4,5)P2: Phosphatidylinositol 4,5-bisphosphate, an anionic phospholipid molecule distributed on the inner side of the cell membrane, creating the cell membrane surface microenvironment and stabilizing membrane proteins.

[0027] DNase I: Deoxyribonuclease I, hydrolyzes deoxyribonucleic acid (DNA).

[0028] In a second aspect, the present invention provides a kit for separating and extracting membrane proteins and cytoplasmic proteins, characterized in that the kit includes the separation and extraction reagents described in the first aspect of the present invention.

[0029] Furthermore, the kit also includes a device for storing the reagents.

[0030] Thirdly, the present invention provides the application of the separation and extraction reagent described in the first aspect of the present invention or the kit described in the second aspect of the present invention in the separation and extraction of membrane proteins and cytoplasmic proteins.

[0031] In some embodiments of the present invention, the application is to separate and extract membrane proteins and cytoplasmic proteins of animal cells, including but not limited to Expi293F cells, CHO-S cells, Sf9 cells or HeLa cells.

[0032] In some embodiments of the present invention, the application is to separate and extract membrane proteins and cytoplasmic proteins from animal tissues, including but not limited to heart tissue.

[0033] In this invention, cytoplasmic proteins refer to cytoplasmic proteins in cells or tissues, and membrane proteins include not only membrane proteins on the plasma membrane, but also membrane proteins on the mitochondrial membrane, endoplasmic reticulum membrane, and Golgi apparatus membrane.

[0034] Fourthly, the present invention provides a method for separating and extracting cell membrane proteins and cytoplasmic proteins, the method comprising the following steps: S1) Resuspend the cells in a centrifuge tube, centrifuge at 300×g for 5 min, and retain the cell pellet; S2) Resuspend the cell pellet in the extraction reagent A described in the first aspect of the present invention into a centrifuge tube, centrifuge at 300×g for 5 min, and retain the cell pellet. S3) Add the extraction reagent B described in the first aspect of the present invention to the cell pellet, mix by pipetting, and then incubate on a rotary mixer for 10 min. S4) Centrifuge the incubated cells at 16,000×g for 15 min, collect the supernatant, which is the cytoplasmic protein, and retain the cell pellet; S5) Add the extraction reagent A described in the first aspect of the present invention to the cell pellet in step S4), mix by pipetting, centrifuge at 16,000×g for 15 min, and retain the cell pellet; S6) Add the extraction reagent C described in the first aspect of the present invention to the cell pellet in step S5), mix by pipetting, and then incubate on a rotary mixer for 45 min. S7) Centrifuge the incubated cells at 16,000×g for 15 min and collect the supernatant, which is the cell membrane protein.

[0035] Further, in step S1), the temperature is room temperature; in step S2), the temperature is room temperature; in step S3), the temperature is 4°C; in step S4), the centrifuge temperature is 4°C; in step S5), the centrifuge temperature is 4°C; in step S6), the temperature is 4°C; in step S7), the centrifuge temperature is 4°C.

[0036] In one specific embodiment of the present invention, the number of cells in step S1) is 5 million; the amount of reagent A used in step S2) is 2 mL; the amount of reagent B used in step S3) is 0.75 mL; the amount of reagent A used in step S5) is 1 mL; and the amount of reagent C used in step S6) is 0.5 mL. The cells are animal cells, including but not limited to Expi293F cells, CHO-S cells, Sf9 cells, or HeLa cells.

[0037] Fifthly, the present invention provides a method for separating and extracting tissue membrane proteins and cytoplasmic proteins, the method comprising the following steps: S1) Place the tissue in a centrifuge tube and wash it with extraction reagent A as described in the first aspect of the present invention; S2) The tissue is cut into small pieces, and the extraction reagent B described in the first aspect of the present invention is added. The mixture is then ground to form a suspension. S3) Continue to add the extraction reagent B described in the first aspect of the present invention, mix by blowing and stirring, and then incubate on a rotary mixer for 10 min; S4) Centrifuge the incubated sample at 16,000×g for 15 min, collect the supernatant, which is the cytoplasmic protein, and retain the precipitate; S5) Add the extraction reagent A described in the first aspect of the present invention to the cell pellet in step S4), mix by pipetting, centrifuge at 16,000×g for 15 min, and retain the cell pellet; S6) Add the extraction reagent C described in the first aspect of the present invention to the precipitate in step S5), mix by blowing and stirring, and then incubate on a rotary mixer for 45 min. S7) Centrifuge the incubated sample at 16,000×g for 15 min, and take the supernatant, which is the membrane protein.

[0038] Further, in step S1), the temperature is room temperature; in step S2), the temperature is 4°C; in step S3), the temperature is 4°C; in step S4), the centrifuge temperature is 4°C; in step S5), the centrifuge temperature is 4°C; in step S6), the temperature is 4°C; in step S7), the centrifuge temperature is 4°C.

[0039] In one specific embodiment of the present invention, in step S1), the tissue weight is 20 mg and the amount of extraction reagent A is 4 mL; in step S2), the amount of extraction reagent B is 1 mL; in step S3), the amount of extraction reagent B is 0.5 mL; in step S5), the amount of extraction reagent A is 1 mL; and in step S6), the amount of extraction reagent C is 1 mL.

[0040] The tissues mentioned are animal tissues, including but not limited to heart tissue.

[0041] The technical solution of this invention has the following beneficial effects: The kit provided by this invention can not only separate and extract membrane proteins and cytoplasmic proteins from animal cells, but also extract membrane proteins from animal tissues. The membrane proteins extracted by the kit include those of the cytoplasmic membrane, organelle membranes, and nuclear membrane. It is more convenient to operate, has a higher yield, and better results than commercially available membrane protein extraction kits, and has promising application prospects.

[0042] Compared with the operational steps of commercially available kits such as Beyotime (P0033), Sanying (PK10015), Yisheng (20127ES50), and Solarbio (EX1110), the kit provided by this invention does not require the use of homogenizers or repeated freeze-thaw cycles to process cells during the separation and extraction of cell membrane proteins and cytoplasmic proteins. The operation is convenient and the process is simple.

[0043] Compared with commercially available kits such as Thermo (89842) and Beyotime (P0033), the kit provided by this invention extracts a higher total amount of membrane proteins. Figure 1 The yield of the target protein is higher. Figure 2 ).

[0044] Compared with commercially available kits such as Thermo (89842), the kit provided by this invention has better separation effect on cell membrane proteins and cytoplasmic proteins, and lower cross-contamination rate. Figure 3 ).

[0045] The cell membrane proteins extracted using the kit provided by this invention have a wide range of applications, not only suitable for qualitative, quantitative, and functional studies of proteins in animal cells, but also for the extraction and study of membrane proteins in animal tissues. Figure 4 ) Attached Figure Description Figure 1 The kits (Epizyme) and Thermo (89842) provided by this invention extract the total amount of membrane proteins from different types of animal cells (BCA quantification method).

[0046] Figure 2 Western blot (WB) results of four overexpressed membrane proteins extracted from Expi293F cells using the kits provided in this invention (Epizyme), Thermo (89842), and Beyotime (P0033).

[0047] Figure 3 The Western blot results of cytoplasmic and cell membrane components extracted from Expi293F cells using the kits (Epizyme) and Thermo (89842) provided in this invention.

[0048] Figure 4 The kit (Epizyme) provided by this invention provides the WB results of membrane proteins and cytoplasmic proteins extracted from 20 mg of mouse heart tissue. Detailed Implementation

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Optimal reagent ratio Extraction reagent A: 20 mM Tris, 150 mM NaCl and 1 mM EDTA.

[0051] Extraction reagent B: 20 ​​mM Tris, 150 mM NaCl, 0.15% (w / v) MβCD, 0.05% (w / v) Digitonion, 1 μg / mL Aprotinin, 2 μg / mL Leupeptin, 2 μg / mL Pepstatin.

[0052] Extraction reagent C: 20 mM Tris, 150 mM NaCl, 1.25% (w / v) LMNG, 0.25% (w / v) GDN, 1mM SCS, 50 μM PI(4,5)P2, 2 μg / mL DNase I, 1 μg / mL Aprotinin, 2 μg / mL Leupeptin, 2 μg / mL Pepstatin.

[0053] The following specific application experiments assume that the separation and extraction reagents used are the reagents with the optimal ratio. The reagent kit formed by this invention is named Epizyme.

[0054] Example 1: Method for the isolation and extraction of adherent / suspension cell membrane proteins (1) Adherent cells: Discard the culture medium in the 6 cm culture dish, add 3 mL of extraction reagent A, scrape the adherent cells from the surface of the culture dish with a cell scraper, and resuspend the cells. Take 5 million cells into a centrifuge tube, centrifuge at 300×g for 5 min, and retain the cell pellet; Suspension cells: Take 5 million cells in suspension culture into a centrifuge tube, centrifuge at 300×g for 5 min, and retain the cell pellet; (2) Resuspend the cell pellet in 1 mL of extraction reagent A, mix by pipetting, centrifuge at 300×g for 5 min, and retain the cell pellet; (3) Add 0.75 mL of extraction reagent B to the cell pellet (obtained in step 2), mix by pipetting, and incubate on a rotary mixer for 10 min. (4) Centrifuge the incubated cells at 16,000×g for 15 min and collect the supernatant, which is the cytoplasmic protein; (5) Add 0.5 mL of extraction reagent C to the cell pellet (obtained in step 4), mix by pipetting, and incubate on a rotary mixer for 45 min. (6) Centrifuge the incubated suspension at 16,000×g for 15 min and take the supernatant, which is the cell membrane protein.

[0055] Experimental group: Following the method provided in Example 1, 5 million suspension cultured Expi293F cells, CHO-S cells, Sf9 cells, and HeLa cells were taken respectively, and membrane proteins were separated and extracted using the kit (Epizyme) provided in this invention. The separated membrane proteins were quantified using the BCA quantification method.

[0056] Control group: Mem-PER™ Plus eukaryotic cell membrane protein extraction kit (catalog number 89842, referred to as Thermo (89842) kit in this invention) was purchased from commercial channels. 5 million suspension cultured Expi293F cells, CHO-S cells, Sf9 cells and HeLa cells were used to extract membrane proteins according to the kit instructions. The isolated membrane proteins were quantified using the BCA quantification method.

[0057] The total amount of membrane proteins obtained by the two extraction methods mentioned above is as follows: Figure 1 As shown, compared with the Thermo (89842) kit, the kit (Epizyme) provided by this invention extracts a higher total amount of membrane proteins.

[0058] Example 2: Method for Isolation and Extraction of Overexpressed Membrane Proteins (1) Four membrane proteins containing the Strep tag (KCNQ2, KCNQ4, KCNQ5 and NTCP, with monomer relative molecular masses of 70.6 kDa, 78.8 kDa, 103.6 kDa and 39.8 kDa, respectively) were overexpressed in 10 mL of Expi293F cells in suspension culture. 5 million cells were collected, centrifuged at 300×g for 5 min, and the cell pellet was retained. (2) Resuspend the cell pellet in 1 mL of extraction reagent A, centrifuge at 300×g for 5 min, and retain the cell pellet; (3) Add 0.75 mL of extraction reagent B to the cell pellet (obtained in step 2), mix by pipetting, and incubate on a rotary mixer for 10 min. (4) Centrifuge the incubated cells at 16,000×g for 15 min and collect the supernatant, which is the cytoplasmic protein; (5) Add 0.5 mL of extraction reagent C to the cell pellet (obtained in step 4), mix by pipetting, and incubate on a rotary mixer for 45 min. (6) Centrifuge the incubated suspension at 16,000×g for 15 min and take the supernatant, which is the cell membrane protein.

[0059] Experimental group: Following the method provided in Example 2, 5 million Expi293F cells overexpressing four membrane proteins in suspension culture were taken, and membrane proteins were separated and extracted using the kit (Epizyme) provided in this invention.

[0060] Control group 1: 5 million suspension cultured Expi293F cells overexpressing 4 membrane proteins were used to extract membrane proteins according to the Thermo (89842) kit instructions.

[0061] Control group 2: The Beyotime Cell Membrane and Cytoplasmic Protein Extraction Kit (product number P0033, referred to as Beyotime (P0033) Kit in this invention) was purchased from commercial channels, and membrane proteins were extracted according to the kit instructions.

[0062] Western blot (WB) was used to detect the membrane proteins extracted by the above method (the final volume of each extracted membrane protein was 500 μL, and the protein loading volume was 10 μL; Strep-tagged antibody was used as the detection indicator). The results are as follows: Figure 2As shown, the target membrane protein bands extracted by the kit (Epizyme) provided by this invention are darker, indicating a higher extraction yield. Gray-scale analysis shows that the amount of target membrane protein extracted by the kit (Epizyme) is 1.89-3.97 times that of the Thermo (89842) kit and 1.62-8.91 times that of the Beyotime (P0033) kit.

[0063] Example 3: An efficient method for separating cell membrane proteins from cytoplasmic proteins (using Expi293F cells as an example). (1) Take 5 million suspension cultured Expi293F cells, centrifuge at 300×g for 5 min, and retain the cell pellet; (2) Resuspend the cell pellet in 1 mL of extraction reagent A, centrifuge at 300×g for 5 min, and retain the cell pellet; (3) Add 0.75 mL of extraction reagent B to the cell pellet (obtained in step 2), mix by pipetting, and incubate on a rotary mixer for 10 min. (4) Centrifuge the incubated cells at 16,000×g for 15 min and collect the supernatant, which is the cytoplasmic protein; (5) Add 1 mL of extraction reagent A to the cell pellet (obtained in step 4), mix well by pipetting, centrifuge at 16,000×g for 15 min, and retain the cell pellet; (6) Add 0.5 mL of extraction reagent C to the cell pellet (obtained in step 5), mix by pipetting, and incubate on a rotary mixer for 45 min. (7) Centrifuge the incubated suspension at 16,000×g for 15 min and take the supernatant, which is the cell membrane protein.

[0064] Experimental group: Following the method provided in Example 3, 5 million suspension cultured Expi293F cells were taken and membrane proteins were separated and extracted using the kit (Epizyme) provided in this invention. Cytoplasmic proteins were obtained from step (4) and membrane proteins were obtained from step (7).

[0065] Control group 1: 5 million suspension cultured Expi293F cells were used to extract membrane proteins according to the Thermo (89842) kit instructions, and cytoplasmic proteins and membrane proteins were obtained respectively.

[0066] Western blot (WB) was used to detect cytoplasmic and membrane proteins extracted by the above method (protein loading volume was 10 μL, and HSP60 / GAPDH antibody was used as the detection index for cytoplasmic proteins). The results are as follows: Figure 3As shown, the Epizyme kit provided by this invention exhibits almost no bands of cytoplasmic proteins in the cell membrane components, while the Thermo (89842) kit shows clearly visible bands of cytoplasmic proteins in the cell membrane components. Grayscale analysis shows that the membrane / plasma cross-contamination rate of the Thermo (89842) kit is approximately 20%, significantly higher than the membrane / plasma cross-contamination rate of the Epizyme kit (<5%).

[0067] Example 4: Method for Isolation and Extraction of Membrane Proteins from Mouse Heart Tissue (1) Place 20 mg of mouse heart tissue in a centrifuge tube, add 4 mL of extraction reagent A for washing, vortex briefly and discard the washing solution, and repeat the washing once; (2) Transfer the tissue to a 2 mL centrifuge tube and cut it into small pieces with scissors. Add 1 mL of extraction reagent B to resuspend the tissue pieces and transfer them to a pre-cooled homogenizer on ice to grind until a homogeneous suspension is formed. (3) Add 0.5 mL of extraction reagent B to the homogenizer, mix by blowing and mixing, transfer to a centrifuge tube, and incubate on a rotary mixer for 10 min. (4) Centrifuge the incubated sample at 16,000×g for 15 min, and take the supernatant, which is the cytoplasmic protein; (5) Add 1 mL of extraction reagent A to the cell pellet (obtained in step 4), mix well by pipetting, centrifuge at 16,000×g for 15 min, and retain the cell pellet; (6) Add 1 mL of extraction reagent C to the precipitate (obtained in step 5), mix well by pipetting, and incubate on a rotary mixer for 45 min; (7) Centrifuge the incubated sample at 16,000×g for 15 min and take the supernatant, which is the cell membrane protein.

[0068] Western blot (WB) was used to detect the cytoplasmic and membrane proteins extracted by the above method (the protein loading volume was 10 μL for each protein, with Caveolin-1 as the indicator for cytoplasmic membrane proteins, COX-IV as the indicator for mitochondrial membrane proteins, and GAPDH as the indicator for cytoplasmic proteins). The results are as follows: Figure 4 As shown, for membrane proteins Caveolin-1 and COX-IV, the bands both appear in the membrane fraction; for cytoplasmic protein GAPDH, the band appears in the cytoplasmic fraction, indicating that the kit (Epizyme) provided by this invention is suitable for the extraction of membrane and cytoplasmic proteins from animal tissues.

[0069] The above specific embodiments are merely illustrative of the invention and do not represent a limitation thereof. Those skilled in the art will recognize that other variations of the specific structure of this invention are possible.

Claims

1. A reagent for separating and extracting membrane proteins and cytoplasmic proteins, characterized in that, The reagent consists of extraction reagent A, extraction reagent B and extraction reagent C; Extraction reagent A includes Tris, NaCl, and EDTA; Extraction reagent B includes Tris, NaCl, MβCD, Digitonion, Aprotinin, Leupeptin, and Pepstatin; Extraction reagent C includes Tris, NaCl, LMNG, GDN, SCS, PI(4,5)P2, DNase I, Aprotinin, Leupeptin, and Pepstatin.

2. The reagent for separating and extracting membrane proteins and cytoplasmic proteins according to claim 1, characterized in that, Extraction reagent A consists of components with the following final concentrations: 10-20 mM Tris, 100-200 mM NaCl and 0.5-1.5 mM EDTA; Extraction reagent B consists of the following components at final concentrations: 10-20 mM Tris, 100-200 mM NaCl, 0.05%-0.15% (w / v) MβCD, 0.05%-0.15% (w / v) Digitonion, 0.5-1 μg / mL Aprotinin, 1.5-2.5 μg / mL Leupeptin, and 1.5-2.5 μg / mL Pepstatin; Extraction reagent C consists of components with the following final concentrations: 10-20 mM Tris, 100-200 mM NaCl, 0.75%-1.25% (w / v) LMNG, 0.25%-0.35% (w / v) GDN, 0.5-1.5 mM SCS, 50-100 μM PI(4,5)P2, 1.5-2 μg / mL DNase I, 0.5-1 μg / mL Aprotinin, 1.5-2.5 μg / mL Leupeptin and 1.5-2.5 μg / mL Epstatin.

3. The reagent for separating and extracting membrane proteins and cytoplasmic proteins according to claim 2, characterized in that, Extraction reagent A consists of the following components at final concentrations: 20 mM Tris, 150 mM NaCl, and 1 mM EDTA; Extraction reagent B consists of the following components at final concentrations: 20 mM Tris, 150 mM NaCl, 0.15% (w / v) MβCD, 0.05% (w / v) Digitonion, 1 μg / mL Aprotinin, 2 μg / mL Leupeptin, and 2 μg / mL Pepstatin. Extraction reagent C consists of the following components at final concentrations: 20 mM Tris, 150 mM NaCl, 1.25% (w / v) LMNG, 0.25% (w / v) GDN, 1 mM SCS, 50 μM PI(4,5)P2, 2 μg / mL DNase I, 1 μg / mL Aprotinin, 2 μg / mL Leupeptin, and 2 μg / mL Pepstatin.

4. A kit for separating and extracting membrane proteins and cytoplasmic proteins, characterized in that, The kit includes the separation and extraction reagents as described in any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes a device for storing the reagents.

6. The use of the separation and extraction reagent according to any one of claims 1-3 or the kit according to any one of claims 4-5 in the separation and extraction of membrane proteins and cytoplasmic proteins; The application is to separate and extract membrane proteins and cytoplasmic proteins from animal cells, including Expi293F cells, CHO-S cells, Sf9 cells, or HeLa cells. The application involves separating and extracting membrane proteins and cytoplasmic proteins from animal tissues, including heart tissue.

7. A method for separating and extracting cell membrane proteins and cytoplasmic proteins, the method comprising the following steps: S1) Resuspend the cells in a centrifuge tube, centrifuge at 300×g for 5 min, and retain the cell pellet; S2) Resuspend the cell pellet in extraction reagent A according to any one of claims 1-3 into a centrifuge tube, centrifuge at 300×g for 5 min, and retain the cell pellet; S3) Add the extraction reagent B according to any one of claims 1-3 to the cell pellet, mix by pipetting, and then incubate on a rotary mixer for 10 min. S4) Centrifuge the incubated cells at 16,000×g for 15 min, collect the supernatant, which is the cytoplasmic protein, and retain the cell pellet; S5) Add the extraction reagent A according to any one of claims 1-3 to the cell pellet of step S4), mix by pipetting, centrifuge at 16,000×g for 15 min, and retain the cell pellet; S6) Add the extraction reagent C according to any one of claims 1-3 to the cell pellet of step S5), mix by pipetting, and incubate on a rotary mixer for 45 min. S7) Centrifuge the incubated cells at 16,000×g for 15 min and collect the supernatant, which is the cell membrane protein.

8. The method according to claim 7, characterized in that, Step S1) Temperature is room temperature; Step S2) Temperature is room temperature; Step S3) Temperature is 4℃; Step S4) Centrifuge temperature is 4℃; Step S5) Centrifuge temperature is 4℃; Step S6) Temperature is 4℃; Step S7) Centrifuge temperature is 4℃.

9. A method for separating and extracting tissue membrane proteins and cytoplasmic proteins, the method comprising the following steps: S1) Place the tissue in a centrifuge tube and wash it with extraction reagent A as described in any one of claims 1-3; S2) The tissue is cut into small pieces, and the extraction reagent B as described in any one of claims 1-3 is added. The mixture is then ground to form a suspension. S3) Continue to add the extraction reagent B according to any one of claims 1-3, mix by pipetting, and then incubate on a rotary mixer for 10 min; S4) Centrifuge the incubated sample at 16,000×g for 15 min, collect the supernatant, which is the cytoplasmic protein, and retain the precipitate; S5) Add the extraction reagent A according to any one of claims 1-3 to the cell pellet of step S4), mix by pipetting, centrifuge at 16,000×g for 15 min, and retain the cell pellet; S6) Add the extraction reagent C according to any one of claims 1-3 to the precipitate in step S5), mix by blowing and stirring, and then incubate on a rotary mixer for 45 min. S7) Centrifuge the incubated sample at 16,000×g for 15 min, and take the supernatant, which is the membrane protein.

10. The method according to claim 9, characterized in that, Step S1) Temperature is room temperature; Step S2) Temperature is 4℃; Step S3) Temperature is 4℃; Step S4) Centrifuge temperature is 4℃; Step S5) Centrifuge temperature is 4℃; Step S6) Temperature is 4℃; Step S7) Centrifuge temperature is 4℃.

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