Lysis solution and its use

Abstract

The application discloses a kind of cracking liquid and its application, the cracking liquid includes group A and group B;Wherein, the group A contains: amino acid buffer, anionic detergent, electrolyte and protective agent;The group B contains: composite enzyme, buffer and cryoprotective agent, the cracking liquid can quickly and completely extract protein in cell and tissue, and effectively protect the chemical modification of protein, can replace existing RIPA type cracking liquid, with good universality, widely used, practical efficient and the like, simultaneously, the cracking liquid is extracted without ultrasonic treatment when protein, not only can the macromolecular protein of molecular weight greater than 90kDa be extracted completely, but also be effective to the protein molecule less than 90kDa application, greatly simplify experimental procedure, save time and material cost, to provide high-quality complete protein raw material for downstream protein analysis at source.

Description

Technical Field

[0001] This invention relates to the field of protein engineering technology, and more specifically, to a lysis buffer and its application. Background Technology

[0002] Proteins are the basic structural and functional units of cells. Proteins vary in molecular weight, and their post-synthesis chemical modifications are diverse and complex. For example, the largest known human protein, titin, has a molecular weight of approximately 3,000 kDa and is composed of about 27,000 amino acids. In contrast, insulin has a molecular weight of only 5.7 kDa and is composed of 51 amino acids. Proteins involved in signal transduction require further chemical modifications after synthesis, collectively known as post-translational modifications. These modifications include phosphorylation, glycosylation, methylation, ubiquitination, acetylation, and biotinylation. Phosphorylation is the most common post-translational modification in cells, a process jointly performed by protein kinases and phosphatases. Upon stimulation by external signals, specific amino acids, such as serine or threonine residues, in protein kinases are phosphorylated. After the stimulation signal ends, phosphatases remove the phosphate groups from the amino acids through hydrolysis.

[0003] Western blotting is a commonly used technique in molecular biology experiments to analyze the expression of a specific protein in cells and tissues. For medium and small-molecule proteins with a molecular weight less than 90 kDa, traditional lysis buffers such as RIPA (Radioimmunoprecipitation Assay Buffer) can effectively dissolve and extract most proteins. Other lysis buffers similar to RIPA include NP-40 lysis buffer, based on the nonionic detergent NP-40 (Nonidet P-40 Substitute). This lysis buffer does not contain sodium dodecyl sulfate (SDS), an anionic detergent that denatures proteins, and is therefore milder than RIPA lysis buffer, primarily used for extracting proteins from the cytoplasm. Another modified version of RIPA lysis buffer is called IP cell lysis buffer (Immunoprecipitation Buffer), which also does not contain SDS and is specifically designed for immunoprecipitation experiments. Regardless of the name of the lysis buffer, these buffer formulations are all derived from RIPA lysis buffer. Furthermore, to prevent protein degradation by the cell's own proteases during extraction, various protease inhibitors are added to the RIPA lysis buffer before cell lysis. To prevent modified signaling proteins from being demodified by the cell's own proteases, various specific enzyme inhibitors are also added. For example, to prevent phosphorylated proteins from being dephosphorylated, phosphatase inhibitors such as sodium fluoride and benzyl sulfonyl fluoride (PMSF) are added to the lysis buffer. Even so, post-translational modified signals cannot be effectively protected.

[0004] However, RIPA and its derivative lysis buffers are not very effective for large protein molecules with a molecular weight greater than 90 kDa. For example, if RIPA lysis buffer is used to lyse proteins in skeletal muscle, large proteins such as titin and myosin will not be lysed and will precipitate. To dissolve large proteins as much as possible, an additional step of sonication is often required in cell and tissue processing. Ultrasound uses physical methods to break down cells and can quickly extract proteins, including large proteins, but the physical action can also break down large protein molecules, making it very difficult to obtain intact large proteins. In addition, because urea is highly soluble in water and can rapidly denature proteins, urea lysis buffer is also often used to extract proteins, including large proteins, from cells and tissues. However, it is well known that urea can carbamate proteins or peptides, ultimately leading to incomplete protein extraction. Other lysis buffers for dissolving large proteins include SDS lysis buffer. This lysis buffer contains up to 1% SDS, but the sonication step is still essential for extracting proteins from cells or tissues.

[0005] Most cell or tissue lysis buffers used in the research market are based on RIPA lysis buffer formulations. These lysis buffers are effective for extracting protein molecules smaller than 90 kDa, but their extraction efficiency is very poor for large proteins larger than 90 kDa. Many laboratories using RIPA lysis buffers to extract large proteins often rely on the physical action of ultrasound to break down cells and aid in the dissolution of large protein molecules. However, ultrasound can also break down large protein peptide chains, resulting in incomplete protein separation. Other chemical agents, such as urea, can help denature and dissolve large proteins, but urea can cause protein formylation, preventing complete protein extraction. To date, commercially available cell or tissue lysis buffers are not effective at extracting large protein molecules. In addition to the above limitations, RIPA lysis buffers have another problem: poor protection against protein chemical modifications such as phosphorylation. Although researchers often add numerous inhibitors to RIPA lysis buffers before cell and tissue lysis to protect against protein chemical modifications, these modifications are easily lost. After repeated freeze-thaw cycles of lysed cells or tissues, some chemical modifications, such as phosphorylation, almost completely disappear.

[0006] In summary, existing cell lysis buffers employ different formulations depending on their intended use to meet the diverse needs of biological experiments. To date, there is still no effective solution for the complete extraction of large protein molecules and the protection of modified proteins during cell lysis.

[0007] Therefore, there is an urgent need to develop a lysis buffer and method for the rapid and complete extraction of macromolecular proteins. Summary of the Invention

[0008] The purpose of this invention is to solve the problems of incomplete extraction of large protein molecules and protection of modified proteins in current cell lysis methods. This invention provides a lysis buffer and its applications. This lysis buffer can rapidly and completely extract proteins from cells and tissues, and effectively protects the chemical modifications of proteins. It can replace existing RIPA-based lysis buffers and has advantages such as good versatility, wide application, practicality, and high efficiency. Furthermore, this lysis buffer does not require ultrasonic treatment during protein extraction. It can completely extract large protein molecules with a molecular weight greater than 90 kDa, and is also effective for protein molecules smaller than 90 kDa, greatly simplifying the experimental process and saving time and material costs. Therefore, it provides high-quality and complete protein raw materials for downstream protein analysis at the source.

[0009] To achieve the above objectives, the first aspect of the present invention provides a lysis buffer comprising formulation A and formulation B; Formula A contains: an amino acid buffer, anionic detergent, electrolyte, and protectant; Formula B contains: a complex enzyme, a buffer solution, and a cryoprotectant.

[0010] A second aspect of the present invention provides the application of the lysis buffer described in the first aspect in protein extraction.

[0011] A third aspect of the present invention provides a method for extracting proteins, the method comprising: 1) The lysis buffer is used to lyse the biological cells or tissue samples treated in the experiment to extract protein samples; 2) Immunoblot analysis of the protein sample was performed to obtain its corresponding expression profile. The pyrolysis solution is the pyrolysis solution described in the first aspect.

[0012] In the above technical solution, the lysis buffer of the present invention (IntactProtein™ Cell Tissue LysisKit, abbreviated as IntactProtein) TM It has the same effect on cell and tissue lysis, can completely extract large protein molecules with a molecular weight greater than 90 kDa from cells or tissues without damaging the protein structure, can well protect the chemical modifications of proteins, and has good versatility, and can also extract proteins with a molecular weight less than 90 kDa.

[0013] Meanwhile, the lysis buffer provided by this invention can extract proteins from tissues or cells intact without ultrasonic treatment, greatly simplifying the experimental process and saving time and material costs, thus providing high-quality and complete protein raw materials for downstream protein analysis at the source.

[0014] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 A shows the expression profile of p-mTOR protein in HeLa cells after starvation for 12 h and stimulation with insulin for 5 min using serum extracted with the lysis buffer prepared in Example 1; B shows the expression profile of mTOR protein in HeLa cells after starvation for 12 h and stimulation with insulin for 5 min using serum extracted with the lysis buffer prepared in Example 1. Figure 2Example 2 shows the expression profiles of mTOR and GAPDH proteins extracted from HeLa cells and mouse brain tissue using the lysis buffer prepared in Example 1. Figure 3 The expression profiles of mTOR protein extracted from HeLa cells using the lysis buffer prepared in Example 1 and the conventional RIPA lysis buffer are shown in Comparative Example 1. Figure 4 Comparative Example 2 shows the expression profiles of O-GlcNAc glycation signals extracted from HeLa cells using the lysis buffer prepared in Example 1 and the conventional RIPA lysis buffer. Figure 5 The expression profiles of SMRT protein extracted from HT1080 cells using the lysis buffer prepared in Example 1 at different treatment times are compared with those of SMRT protein extracted from HT1080 cells using conventional RIPA lysis buffer combined with ultrasonic treatment in Comparative Example 3. Detailed Implementation

[0016] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] The first aspect of the present invention provides a lysis buffer comprising formulation A and formulation B; Formula A contains: an amino acid buffer, anionic detergent, electrolyte, and protectant; Formula B contains: a complex enzyme, a buffer solution, and a cryoprotectant.

[0019] The lysis buffer of this invention has a good lysis effect on cells and tissues, and can extract proteins from cells or tissues intact without damaging the protein structure and protecting the chemical modifications of the proteins. The lysis buffer can also extract proteins of different molecular weights and has good versatility.

[0020] In a preferred embodiment of the present invention, the amino acid buffer is selected from one or more of glycine, tris(hydroxymethyl)aminomethane, glutamic acid and histidine.

[0021] In a preferred embodiment of the present invention, the amino acid buffer is glycine or tris(hydroxymethyl)aminomethane.

[0022] In a preferred embodiment of the present invention, in order to achieve better lysis performance of the lysis buffer, the concentration of the amino acid buffer is 12-25 g / L.

[0023] In a preferred embodiment of the present invention, the anionic detergent is selected from one or more of sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, sodium taurocholate, and bile acids.

[0024] In a preferred embodiment of the present invention, the anionic detergent is selected from one or more of sodium dodecyl sulfate, sodium cholate, or sodium deoxycholate.

[0025] In a preferred embodiment of the present invention, in order to achieve better pyrolysis effect of the lysis solution, the concentration of the anionic detergent is 15-35 g / L.

[0026] In a preferred embodiment of the present invention, the electrolyte is selected from two or more of sodium chloride, magnesium chloride, magnesium sulfate, and potassium chloride.

[0027] In a preferred embodiment of the present invention, the electrolyte is selected from two or more of sodium chloride, magnesium chloride and magnesium sulfate.

[0028] In a preferred embodiment of the present invention, in order to achieve better lysis performance of the lysis solution, the concentration of the electrolyte is 0.2-0.8 g / L.

[0029] In a preferred embodiment of the present invention, the protective agent is selected from one or more of glycerol, dimethyl sulfoxide, and albumin.

[0030] In a preferred embodiment of the present invention, the protective agent is selected from glycerol or albumin.

[0031] In a preferred embodiment of the present invention, in order to achieve better pyrolysis effect of the lysis solution, the concentration of the protective agent is 60-260 g / L.

[0032] In a preferred embodiment of the present invention, the formulation A further contains a solvent.

[0033] Preferably, the solvent is water.

[0034] In a preferred embodiment of the present invention, the complex enzyme includes RNase, DNase, endonuclease and exonuclease.

[0035] In a preferred embodiment of the present invention, the volume ratio of the RNase, DNase, endonuclease and exonuclease is 1-2:1-2:1-2:1-2, for example, 2:1:1:1, 1:1:1:2, 1:1:1:1.

[0036] Preferably, the volume ratio of the RNase, DNase, endonuclease, and exonuclease is 1:1:1:1.

[0037] In a preferred embodiment of the present invention, the enzyme activity in formulation B is 20-75 U / µL, preferably 55 U / µL, and can also be 20 U / µL, 60 U / µL, 65 U / µL or 75 U / µL.

[0038] In a preferred embodiment of the present invention, the buffer solution is selected from one or more of potassium phosphate buffer, citrate buffer, and tris(hydroxymethyl)aminomethane buffer.

[0039] In a preferred embodiment of the present invention, the pH of the buffer solution is 7.8-8.8, preferably 8.2.

[0040] In a preferred embodiment of the present invention, the cryoprotectant comprises a polyol.

[0041] In a preferred embodiment of the present invention, the polyol is selected from one or more of glycerol, sorbitol and mannitol.

[0042] In a preferred embodiment of the present invention, the preparation method of the formulation A includes: mixing an amino acid buffer, an anionic detergent, an electrolyte, a protective agent and a solvent, and adjusting the pH to 7.8-8.8.

[0043] In a preferred embodiment of the present invention, the mixing conditions in the preparation method of formulation A include: a time of 10-60 min, preferably 20 min, and also 10 min, 15 min, 30 min, 40 min, 50 min, or 60 min; and a temperature of 15-30°C, preferably 25°C.

[0044] In a preferred embodiment of the present invention, the pH value in the preparation method of formulation A is 8.5.

[0045] In a preferred embodiment of the present invention, the preparation method of formulation B includes: first mixing the buffer solution and the cryoprotectant, and then adding the complex enzyme for a second mixing.

[0046] In a preferred embodiment of the present invention, in the preparation method of formulation B, the conditions for the first mixing include: the volume ratio of the buffer solution and the cryoprotectant is 1-2:1-2, preferably 1:1, and may also be 2:1, 1:2 or 1.5:1.

[0047] In a preferred embodiment of the present invention, the complex enzyme is prepared by RNase, DNase, endonuclease and exonuclease in a volume ratio of 1-2:1-2:1-2:1-2.

[0048] Preferably, the volume ratio of RNase, DNase, endonuclease, and exonuclease is 1:1:1:1.

[0049] In a preferred embodiment of the present invention, the preparation method of formulation B includes the following conditions for the second mixing: time of 5-30 min, preferably 10 min; and temperature of 0-25℃, preferably 4℃.

[0050] Formula B in this invention needs to be stored at -20°C or below.

[0051] A second aspect of the present invention provides the application of the lysis buffer described in the first aspect in protein extraction.

[0052] A third aspect of the present invention provides a method for extracting proteins, the method comprising: 1) The lysis buffer is used to lyse the biological cells or tissue samples treated in the experiment to extract protein samples; 2) Immunoblot analysis of the protein sample was performed to obtain its corresponding expression profile. The pyrolysis solution is either the pyrolysis solution described in the first aspect or the pyrolysis solution described in the third aspect.

[0053] The protein extraction method provided by this invention does not require ultrasonic treatment, can completely extract proteins of different molecular weights, simplifies the experimental process, and saves time and material costs, providing high-quality and complete protein raw materials for downstream protein analysis at the source.

[0054] In a preferred embodiment of the present invention, in order to make cell or tissue lysis more rapid and protein samples extracted more quickly, the lysis conditions in step 1) include: lysis is carried out in ice or ice water for 10-20 minutes.

[0055] In a preferred embodiment of the present invention, in order to obtain the protein expression profile of the extracted protein sample more quickly and completely, the conditions for the immunoblotting analysis in step 2) include: adding a reducing agent and an indicator to the protein sample, heating the protein sample, and finally performing SDS-PAGE to obtain the protein expression profile of the protein sample.

[0056] In a preferred embodiment of the present invention, the type of reducing agent can be selected from a wide range, and can be a reducing agent conventionally used in the art, such as β-mercaptoethanol with a mass fraction of 2-5% or 50 mMDTT with a molar concentration.

[0057] In a preferred embodiment of the present invention, the type of indicator can be selected from a wide range and can be an indicator conventionally used in the art, such as bromophenol blue with a mass fraction of 0.1%.

[0058] In a preferred embodiment of the present invention, the heating conditions include: a temperature of 95-100°C and a time of 2-8 min.

[0059] In a preferred embodiment of the present invention, in order to ensure that the prepared lysis solution has a better lysis effect, the lysis solution needs to be prepared and used immediately, wherein the volume ratio of formulation A to formulation B is 250-2000:1-5.

[0060] Preferably, the volume ratio of formulation A to formulation B is 500:1.

[0061] In a preferred embodiment of the present invention, in order to ensure that the prepared lysis solution has a better lysis effect, the mixing conditions of formulation A and formulation B include: time of 5-15 min and temperature of 0-25℃.

[0062] The present invention will be described in detail below through examples, but the scope of protection of the present invention is not limited thereto. In the following examples, the drugs and pharmaceuticals are all conventional commercially available products.

[0063] Example 1 (1) Preparation of 1000mL Formula A: Add 900mL of distilled water to a clean beaker, add 18g of glycine, 25g of sodium dodecyl sulfate, 100mL (129.7g) of glycerol, 0.3g of magnesium chloride and 0.2g of sodium chloride, adjust the pH of the mixed solution to 8.5, and make up to 1000mL with distilled water. Mix thoroughly for 20min and store at 25℃ for later use.

[0064] (2) Preparation of 100 mL of formulation B: Prepare 50 mL of 10 mM tris(hydroxymethyl)aminomethane aqueous solution, adjust the pH to 8.2, add glycerol to the final concentration of 50% (v / v), vortex mix, add complex enzyme (RNase, DNase, endonuclease and exonuclease in a volume ratio of 1:1:1:1) to make the final enzyme activity 55 U / µL, mix thoroughly at 4℃ for 10 min, dispense into small tubes, and store at -20℃ for later use.

[0065] (3) Before use, immediately add 10 µL of formulation B to 5 mL of formulation A, vortex for 5 min to thoroughly mix the reagents in formulation A and formulation B, and prepare the lysis buffer IntactProtein. TM And place it on ice.

[0066] Example 2 (1) Preparation of 1000mL Formula A: Add 900mL of distilled water to a clean beaker, add 12g of glycine, 15g of sodium dodecyl sulfate, 50mL (64.85g) of glycerol, 0.1g of magnesium chloride and 0.1g of sodium chloride, adjust the pH of the mixed solution to 7.8, and make up to 1000mL with distilled water. Mix thoroughly for 10min and store at 15℃ for later use.

[0067] (2) Preparation of 100 mL of formulation B: Prepare 25 mL of 10 mM tris(hydroxymethyl)aminomethane aqueous solution, adjust the pH to 7.8, add glycerol to the final concentration of 25% (v / v), vortex mix, add complex enzyme (RNase, DNase, endonuclease and exonuclease in a volume ratio of 1:1:1:1) to make the final enzyme activity 20 U / µL, mix thoroughly at 4℃ for 5 min, dispense into small tubes, and store at -20℃ for later use.

[0068] (3) Before use, immediately add 10 µL of formulation B to 5 mL of formulation A, vortex for 5 min to thoroughly mix the reagents in formulation A and formulation B, and prepare the lysis buffer IntactProtein. TM And place it on ice.

[0069] Example 3 (1) Preparation of 1000mL Formula A: Add 900 mL of distilled water to a clean beaker, add 25 g of glycine, 35 g of sodium dodecyl sulfate, 100 mL (259.4 g) of glycerol, 0.4 g of magnesium chloride and 0.4 g of sodium chloride, adjust the pH of the mixed solution to 8.8, and make up to 1000 mL with distilled water. Mix thoroughly for 60 min and store at 30℃ for later use.

[0070] (2) Preparation of 100 mL of formulation B: Prepare 75 mL of 10 mM tris(hydroxymethyl)aminomethane aqueous solution, adjust the pH to 8.2, add glycerol to the final concentration of 75% (v / v), vortex mix, add complex enzyme (RNase, DNase, endonuclease and exonuclease in a volume ratio of 1:1:1:1) to make the final enzyme activity 75 U / µL, mix thoroughly at 4℃ for 30 min, dispense into small tubes, and store at -20℃ for later use.

[0071] (3) Before use, immediately add 10 µL of formulation B to 5 mL of formulation A, vortex for 5 min to thoroughly mix the reagents in formulation A and formulation B, and prepare the lysis buffer IntactProtein. TM And place it on ice.

[0072] Application Example 1 After the experimental treatment, the adherent HeLa cells cultured in the experiment were collected, the culture medium was removed, and the cells were washed twice with ice-cold PBS (2-8°C). Place the culture dish / plate containing HeLa cells on ice and press 5×10 6 Add the IntactProtein lysis buffer prepared in Example 1 to the culture dish at a ratio of 1 mL per cell. TM Mix HeLa cells and lysis buffer using a pipette and lyse on ice for 5 min; Then, using a clean plastic scraper, scrape the adherent HeLa cells from the culture dish / plate, collect the cell lysis products into a 1.5 mL centrifuge tube, vortex for 30 seconds to mix the lysis products, and place the lysis products on ice for 10 minutes to complete the lysis. The cell lysis products were then heated at 95°C for 5 min, cooled on ice for 3 min, centrifuged at 13,000 g for 5 min at 4°C, and the protein concentration was measured using a spectrophotometer. The lysis products were aliquoted and stored at -20°C for later use. SDS-PAGE gel electrophoresis: The lysate was heated at 95°C for 5 min, then cooled on ice for 3 min, centrifuged at 13,000 g for 5 min at 4°C, and then β-mercaptoethanol (3% final concentration) and bromophenol blue (0.1% added) were added to the lysate for SDS-PAGE. The mixture was then transferred to a membrane and incubated overnight at 4°C with primary and secondary antibodies to amplify the immunoblot signal. The membrane was then exposed to light to obtain the expression profile of the protein.

[0073] Application Example 2 After the experimental treatment, the Raji cells in suspension culture were collected, centrifuged at 300 g for 5 min, and then resuspended in 10 mL of ice-cold PBS (2-8°C). After centrifugation again, the PBS was discarded, and the cells were resuspended in the remaining PBS using a pipette. Place centrifuge tubes containing Raji cells on ice and press 5 × 10⁻⁶ cells / mL. 6 Add the IntactProtein lysis buffer prepared in Example 1 to the centrifuge tube at a ratio of 1 mL per cell. TM Mix Raji cells and lysis buffer with a pipette and lyse on ice for 5 min. Collect the cell lysis products into a 1.5 ml centrifuge tube, vortex for 30 seconds to mix the lysis products, and place the lysis products on ice for another 10 minutes to complete the lysis. Subsequently, the cell lysis products were heated at 95°C for 5 min, then cooled at 13,000 g for 3 min, and centrifuged at 4°C for 5 min. The protein concentration was measured using a spectrophotometer. The lysis products were aliquoted and stored at -20°C for later use. SDS-PAGE gel electrophoresis: The lysate was heated at 95°C for 5 min, then cooled on ice for 3 min, centrifuged at 13,000 g for 5 min at 4°C, and then β-mercaptoethanol (3% final concentration) and bromophenol blue (0.1% added) were added to the lysate for SDS-PAGE. The mixture was then transferred to a membrane and incubated overnight at 4°C with primary and secondary antibodies to amplify the immunoblot signal. The membrane was then exposed to light to obtain the expression profile of the protein.

[0074] Application Example 3 In liquid nitrogen, mouse brain tissue was ground into fine particles using a mortar and pestle. The IntactProtein lysis buffer prepared in Example 1 was added to the mouse brain tissue at a ratio of 1 g of frozen tissue particles to 3 mL of lysis buffer. TM Then, the homogenized sample was placed on ice for more than 15 min to completely lyse the mouse brain tissue (Note: If there are multiple samples, all homogenized samples should be placed on ice until the last sample is completed), and then centrifuged at 13,000 g for 10 min at 4 °C. Transfer the supernatant containing protein to a 1.5 ml centrifuge tube, vortex for 30 seconds to mix the lysis product, and place the lysis product on ice for another 10 minutes to complete the lysis. Subsequently, the cell lysis products were heated at 95°C for 5 min, then cooled at 13,000 g for 3 min, and centrifuged at 4°C for 5 min. The protein concentration was measured using a spectrophotometer. The lysis products were aliquoted and stored at -20°C for later use. SDS-PAGE gel electrophoresis: The lysate was heated at 95°C for 5 min, then cooled on ice for 3 min, centrifuged at 13,000 g for 5 min at 4°C, and then β-mercaptoethanol (3% final concentration) and bromophenol blue (0.1% added) were added to the lysate for SDS-PAGE. The mixture was then transferred to a membrane and incubated overnight at 4°C with primary and secondary antibodies to amplify the immunoblot signal. The membrane was then exposed to light to obtain the expression profile of the protein.

[0075] Application Example 4 The procedure was performed in the same manner as in Application Example 1, except that HeLa cells were replaced with SH-SY5Y cells.

[0076] Application Example 5 The procedure was performed in the same manner as in Application Example 1, except that HeLa cells were replaced with C2C12 cells.

[0077] Application Example 6 The procedure was performed in the same manner as in Application Example 2, except that Raji cells were replaced with Daudi cells.

[0078] Application Example 7 The procedure was performed in the same manner as in Application Example 2, except that Raji cells were replaced with Jurkat cells.

[0079] Application Example 8 The procedure was performed in the same manner as in Application Example 3, except that the mouse brain tissue was replaced with mouse heart tissue.

[0080] Application Example 9 The procedure was performed in the same manner as in Application Example 3, except that the mouse brain tissue was replaced with mouse muscle tissue.

[0081] Application Example 10 Following the operating procedure of Application Example 1, the IntactProtein lysis buffer prepared in Example 1 was used. TM The expression status of phosphorylated mTOR (p-mTOR) and total mTOR protein (289 kDa) was extracted from HeLa cells after 12 h of serum starvation and 5 min of insulin stimulation, respectively. The results are shown in [Figure number missing]. Figure 1 AB.

[0082] Depend on Figure 1Results A and B show that the lysis buffer prepared in Example 1 of this invention can effectively extract p-mTOR protein and total mTOR protein from HeLa cells after 12 h of serum starvation and 5 min of insulin stimulation. The expression profiles of these proteins show differential characteristics, indicating that the lysis buffer of this invention still exhibits excellent lysis ability for cells under different treatment conditions and has good stability and practicality.

[0083] Application Example 11 Following the operating methods of Application Example 1 and Application Example 3 respectively, the lysis buffer IntactProtein prepared in Example 1 was used. TM Proteins were extracted from adherent HeLa cells and mouse brain tissue, respectively, and analyzed using Western blotting. The results are as follows: Figure 2 As shown.

[0084] Depend on Figure 2 As can be seen from A, the lysis buffer of this invention successfully extracted 289 kDa mTOR protein from adherent HeLa cells and mouse brain tissue; from Figure 2 As can be seen from B, the lysis buffer of the present invention successfully extracted 37 kDa GAPDH protein from adherent HeLa cells and mouse brain tissue, indicating that the lysis buffer of the present invention can not only extract large molecular proteins such as 289 kDa mTOR protein from cells and tissues, but is also very effective in extracting small molecular proteins such as 37 kDa GAPDH protein.

[0085] Comparative Example 1 Following the method of Application Example 1, the lysis buffer IntactProtein prepared in Example 1 was used. TM The results were compared with those obtained by immunoblotting analysis of 289 kDa mTOR protein extracted from adherent HeLa cells using traditional RIPA lysis buffer. Figure 3 As shown.

[0086] Depend on Figure 3 It can be seen that the band of 289 kDa mTOR protein extracted from adherent HeLa cells by the lysis buffer prepared in this invention is clear, while the band of 289 kDa mTOR protein extracted from adherent HeLa cells by the traditional RIPA lysis buffer is light in color and blurry. This indicates that the lysis buffer prepared in this invention has a very good extraction effect on 289 kDa mTOR protein from HeLa cells.

[0087] Comparative Example 2 Following the operating method of Application Example 1, the IntactProtein lysis buffer prepared in Example 1 was used. TM O-GlcNAc glycosylated protein signals were extracted from adherent HeLa cells using both RIPA lysis buffer and conventional RIPA lysis buffer. The results are as follows: Figure 4As shown.

[0088] Depend on Figure 4 It is known that the O-GlcNAc glycosylated protein signal extracted from HeLa cells using traditional RIPA lysis buffer is light in color and not obvious, while the O-GlcNAc glycosylated protein signal extracted from HeLa cells using this application is dark in color and the bands are clear, indicating that the lysis buffer prepared in this invention can effectively protect the modified protein from degradation.

[0089] Comparative Example 3 Following the operating method of Application Example 1, the IntactProtein lysis buffer prepared in Example 1 was used. TM After treating HT1080 cells for 5 min, 15 min, and 30 min respectively, lysis products were extracted, and the 270 / 160 kDa SMRT protein in HT1080 cells was detected by immunoblotting. Alternatively, HT1080 cells were treated with conventional RIPA lysis buffer combined with sonication, and then lysis products were extracted. The 270 / 160 kDa SMRT protein in HT1080 cells was detected by Western blotting. The results are as follows: Figure 5 As shown.

[0090] Depend on Figure 5 It can be seen that the expression spectrum of SMRT protein in the lysis product obtained by lysis with the lysis buffer prepared in Example 1 of the present invention is clear, while the expression spectrum of SMRT protein in the lysis product obtained by conventional RIPA lysis buffer combined with ultrasonic treatment shows that ultrasonic treatment breaks the SMRT protein into polypeptide chain.

[0091] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0092] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0093] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A lysis buffer, characterized in that, The lysis buffer comprises formulation A and formulation B; Formula A contains: an amino acid buffer, anionic detergent, electrolyte, and protectant; Formula B contains: a complex enzyme, a buffer solution, and a cryoprotectant; The amino acid buffer is glycine, and the concentration of the amino acid buffer is 12-25 g / L; The anionic detergent is sodium dodecyl sulfate, and the concentration of the anionic detergent is 15-35 g / L; the protective agent is glycerin, and the concentration of the protective agent is 60-260 g / L. The pH of the buffer solution is 7.8-8.8; The complex enzyme includes RNase, DNase, endonuclease, and exonuclease; the buffer solution is tris(hydroxymethyl)aminomethane buffer; and the cryoprotectant is glycerol.

2. The pyrolysis solution according to claim 1, characterized in that, The electrolyte is selected from two or more of sodium chloride, magnesium chloride, magnesium sulfate, and potassium chloride; And / or, the concentration of the electrolyte is 0.2-0.8 g / L; And / or, the formulation A may also contain a solvent.

3. The pyrolysis solution according to claim 2, characterized in that, The electrolyte is selected from two or more of sodium chloride, magnesium chloride, and magnesium sulfate.

4. The pyrolysis solution according to claim 1, characterized in that, In the formulation B, the volume ratio of RNase, DNase, endonuclease and exonuclease is 1-2:1-2:1-2:1-2; And / or, the enzyme activity in formulation B is 20-75 U / µL.

5. The pyrolysis solution according to claim 4, characterized in that, The volume ratio of the RNase, DNase, endonuclease, and exonuclease is 1:1:1:1; And / or, the enzyme activity in formulation B is 55 U / µL.

6. The pyrolysis solution according to claim 2, characterized in that, The preparation method of the formulation A includes: mixing an amino acid buffer, anionic detergent, electrolyte, protective agent and solvent, and adjusting the pH to 7.8-8.

8.

7. The pyrolysis solution according to claim 6, characterized in that, The mixing conditions include: a time of 10-60 minutes and a temperature of 15-30°C.

8. The pyrolysis solution according to claim 7, characterized in that, The time is 20 minutes; And / or, the temperature is 25°C; And / or, the pH is 8.

5.

9. The pyrolysis solution according to claim 1, characterized in that, The preparation method of formulation B includes: first mixing the buffer solution and the cryoprotectant, and then adding the complex enzyme for a second mixing.

10. The pyrolysis solution according to claim 9, characterized in that, The conditions for the first mixing include a volume ratio of the buffer solution to the cryoprotectant of 1-2:1-2.

11. The pyrolysis solution according to claim 10, characterized in that, The volume ratio of the buffer solution to the cryoprotectant is 1:

1.

12. The pyrolysis solution according to claim 9, characterized in that, The conditions for the second mixing include: a time of 5-30 minutes and a temperature of 0-25°C.

13. The lysis buffer according to claim 12, characterized in that, The time is 10 minutes; And / or, the temperature is 4°C.

14. The use of a lysis buffer as described in any one of claims 1-13 in protein extraction.

15. A method for extracting proteins, characterized in that, The method includes: 1) The lysis buffer is used to lyse the biological cells or tissue samples treated in the experiment to extract protein samples; 2) Immunoblot analysis of the protein sample was performed to obtain its corresponding expression profile. The pyrolysis solution is any one of the pyrolysis solutions according to claims 1-13.

16. The method according to claim 15, characterized in that, In step 1), the pyrolysis conditions include: being carried out in ice and / or ice water for 10-20 minutes.

17. The method according to claim 15, characterized in that, In step 2), the conditions for the immunoblotting analysis include: adding a reducing agent and an indicator to the protein sample, heating the protein sample, and finally performing SDS-PAGE to obtain the protein expression profile of the protein sample.

18. The method according to claim 17, characterized in that, The reducing agent is selected from β-mercaptoethanol with a mass fraction of 2-5% and / or DTT with a molar concentration of 50 mM; And / or, the indicator is bromophenol blue with a mass fraction of 0.1%; And / or, the heating conditions include: a temperature of 95-100°C and a time of 2-8 min.

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