Phosphorylated protein separation electrophoresis prefabricated glue capable of being stored for long time

Abstract

The invention relates to the technical field of biomacromolecule detection, and discloses a long-term preserved prefabricated gel for phosphorylated protein separation electrophoresis, the long-term preserved prefabricated gel comprises a polyacrylamide prefabricated gel, the polyacrylamide prefabricated gel comprises a binuclear metal complex capable of being specifically combined with a phosphorylated group, the binuclear metal complex is a compound T67772, and the binuclear metal complex is a compound T67772. Divalent metal ions are added into the polyacrylamide prefabricated glue, and the divalent metal ions are zinc ions or manganese ions. A binuclear metal complex (a compound T67772) and divalent metal ions (such as zinc ions or manganese ions) are added into prefabricated glue, and the prefabricated glue can be specifically combined with phosphate groups in phosphorylated proteins, so that the mobility of the phosphorylated proteins is remarkably reduced, effective separation from non-phosphorylated proteins is realized, and the yield of the phosphorylated proteins is improved. The specific binding mode ensures the migration difference of phosphorylated and non-phosphorylated proteins, and realizes the separation of protein subtypes with different phosphorylation numbers and sites.

Description

Technical Field

[0001] The invention relates to the technical field of biomacromolecule detection, in particular to a precast gel for phosphorylated protein separation electrophoresis which can be stored for a long time. Background Art

[0002] Most regulatory mechanisms of proteins are mediated by conformational changes of proteins, which are usually mediated by allosteric effects and various covalent modifications on the primary structure of proteins. The reversible reaction of protein phosphorylation and dephosphorylation is one of the most common and important post-translational modifications in the body. It involves physiological processes such as cell signaling, cell differentiation and growth, cell metabolism and apoptosis. In addition, this phosphorylation and dephosphorylation plays an important role in disease-related metabolic systems, and abnormal phosphorylation and dephosphorylation are one of the causes of cell carcinogenesis. Exploring the phosphorylated-nonphosphorylated state of proteins is not only conducive to the study of gene expression and enzyme activity in tissue cells, but also helpful for the diagnosis and treatment of diseases.

[0003] However, traditional precast gels can only separate proteins according to their molecular weight and cannot distinguish between phosphorylated and non-phosphorylated proteins, which to some extent limits the efficiency and separation effect of phosphorylated protein detection. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a precast gel for electrophoresis of phosphorylated protein separation that can be stored for a long time, which solves the problem that traditional precast gels can only separate proteins according to their molecular weight and cannot distinguish between phosphorylated and non-phosphorylated proteins.

[0005] To achieve the above objectives, the present invention is implemented through the following technical scheme: a precast gel for phosphorylated protein separation electrophoresis that can be stored for a long time, including a polyacrylamide precast gel, the polyacrylamide precast gel contains a binuclear metal complex that can specifically bind to phosphorylated groups, the binuclear metal complex is compound T67772, and the polyacrylamide precast gel is added with divalent metal ions, and the divalent metal ions are zinc ions or manganese ions.

[0006] Preferably, the concentration of the divalent metal ions in the polyacrylamide precast gel is twice that of compound T67772.

[0007] Preferably, the concentration of the compound T67772 in the polyacrylamide precast gel is in the range of 10 to 300 micromolar.

[0008] Preferably, the polyacrylamide precast gel consists of an upper layer of concentrated gel and a lower layer of separation gel, and the compound T67772 and the divalent metal ions are added to the lower layer of separation gel.

[0009] Preferably, a method for preparing a precast gel for phosphorylated protein separation electrophoresis that can be stored for a long time comprises the following steps:

[0010] S1. Prepare acrylamide solution by dissolving acrylamide powder and methylenebisacrylamide powder in ultrapure water, dilute to 100 ml, and filter for later use;

[0011] S2, prepare separation gel, mix acrylamide solution, buffer system, binuclear metal complex solution, divalent metal ion solution, ultrapure water and ammonium persulfate solution, add tetramethylethylenediamine just before pouring the gel, mix well, pour the gel quickly to obtain separation gel, add ultrapure water pressure gel on the upper layer of the separation gel, and remove it after solidification;

[0012] S3. Prepare concentrated gel. Mix acrylamide solution, buffer system, ultrapure water and ammonium persulfate solution. Add tetramethylethylenediamine just before pouring the gel. Mix well. Quickly pour the gel to obtain concentrated gel. Insert a comb into the concentrated gel and remove it after solidification.

[0013] Preferably, in the step S1, 28-30 g of acrylamide powder and 0.8-1.2 g of methylene bisacrylamide powder are weighed, dissolved in ultrapure water, fixed to 100 ml, and filtered through a 0.45-0.50 μm filter membrane for later use.

[0014] Preferably, in the step S2, 3-3.5 ml of acrylamide solution, 0.8-1.2 ml of buffer system, 150-250 μl of 5 mmol compound T67772 solution, 150-250 μl of 10 mmol ZnCl2 solution, 5-5.5 ml of ultrapure water and 60-80 μl of ammonium persulfate solution are mixed, 4-6 μl of tetramethylethylenediamine is added before pouring the glue, the mixture is evenly mixed, the glue is poured quickly, ultrapure water is added after pouring the glue to press the glue, and the ultrapure water on the upper layer of the separation gel is removed after solidification.

[0015] Preferably, in the step S3, 0.5-0.7 ml of acrylamide solution, 0.3-0.5 ml of buffer system, 2.8-3.0 ml of ultrapure water and 25-35 μl of 10% ammonium persulfate solution are taken, 1-3 μl of tetramethylethylenediamine is added just before pouring the glue, the mixture is mixed evenly, the glue is poured quickly, a comb is inserted into the concentrated glue after pouring the glue, and the precast glue is obtained after solidification, which is stored in the buffer system solution and refrigerated at a temperature of 2 to 8°C.

[0016] Preferably, the buffer system is tris(hydroxymethyl)aminomethane-glycine, bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane) or HEPES-tris(hydroxymethyl)aminomethane, and the pH of the buffer system is neutral.

[0017] The invention provides a precast gel for phosphorylated protein separation electrophoresis which can be stored for a long time.

[0018] It has the following beneficial effects:

[0019] 1. The present invention adds a binuclear metal complex (compound T67772) and a divalent metal ion (such as a zinc ion or a manganese ion) to the precast gel. The precast gel can specifically bind to the phosphate groups in the phosphorylated protein, significantly reduce the mobility of the phosphorylated protein, and thus effectively separate it from the non-phosphorylated protein. This specific binding method ensures the migration difference between the phosphorylated and non-phosphorylated proteins, and realizes the separation of protein subtypes with different phosphorylation numbers and sites.

[0020] 2. The present invention does not require the use of radioisotope-labeled or phosphorylated antibodies for detection, thus avoiding the risk to the health of experimenters and the high cost of antibodies, and reducing the complexity of the operation.

[0021] 3. The precast gel of the present invention can maintain stability for up to 12 months after refrigeration, avoiding the need for frequent preparation of gel, facilitating laboratory management, and reducing experimental costs and time investment.

[0022] 4. The precast gel of the present invention can be directly used in conventional SDS-PAGE electrophoresis operations without the need for additional complex pretreatment steps. The pH value of the buffer system is neutral, which helps to provide a stable electrophoresis environment under denaturing conditions. The neutral pH reduces the degradation rate of the three-dimensional network structure of polyacrylamide in the precast gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the molecular formula of compound T67772 of the present invention;

[0024] Figure 2 The original electrophoresis image of Example 6 of the present invention;

[0025] Figure 3 The original electrophoresis image in Comparative Example 1 of the present invention;

[0026] Figure 4 A schematic diagram showing the comparison of Comparative Example 1 and Example 6 of the present invention and the change in the mobility of phosphorylated proteins;

[0027] Figure 5 This is the electrophoresis diagram of the precast gel in Example 9 of the present invention stored in a refrigerator at 2-8°C for 12 months. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Please see attached Figure 1 - Attachment Figure 5 An embodiment of the present invention provides a precast gel for phosphorylated protein separation electrophoresis that can be stored for a long time, including a polyacrylamide precast gel, the polyacrylamide precast gel contains a binuclear metal complex that can specifically bind to phosphorylated groups, the binuclear metal complex is compound T67772, and the polyacrylamide precast gel is added with divalent metal ions, and the divalent metal ions are zinc ions or manganese ions.

[0030] The concentration of divalent metal ions in the polyacrylamide precast gel is twice that of compound T67772.

[0031] Specifically, the binuclear metal complex contained in the polyacrylamide precast gel is compound T67772 (Phos-tagAcrylamideAAL-107), which can specifically bind to the phosphate groups in the protein in the presence of divalent metal ions. One molecule of compound T67772 can bind to two divalent metal ions, so the concentration of divalent metal ions in the gel is twice that of compound T67772.

[0032] The complex of divalent metal ion-compound T67772 can bind to the phosphate group in a 1:1 ratio, thereby reducing the mobility of phosphorylated proteins in the electric field, thereby achieving effective separation from non-phosphorylated proteins.

[0033] The concentration of compound T67772 in the precast polyacrylamide gel ranged from 10 to 300 micromolar.

[0034] Specifically, the concentration range of compound T67772 in the polyacrylamide precast gel is 10 to 300 micromolar, preferably 20 to 150 micromolar.

[0035] By adjusting the concentration of compound T67772, the separation of phosphorylated proteins can be optimized to accommodate changes in the number of phosphate groups in different samples.

[0036] The polyacrylamide precast gel consists of an upper layer of concentrated gel and a lower layer of separation gel, and compound T67772 and divalent metal ions are added to the lower layer of separation gel.

[0037] Specifically, the polyacrylamide precast gel is composed of an upper concentrated gel and a lower separation gel. Compound T67772 and divalent metal ions are added to the lower separation gel.

[0038] The stacking gel is used to concentrate and initially separate the sample, while the separation gel performs specific separation by binding to phosphorylation groups. This layered structure allows the sample to be concentrated in the stacking gel first and then enter the separation gel during electrophoresis, thereby achieving higher resolution protein separation.

[0039] A method for preparing a precast gel for phosphorylated protein separation electrophoresis that can be stored for a long time, comprising the following steps:

[0040] S1. Prepare acrylamide solution by dissolving acrylamide powder and methylenebisacrylamide powder in ultrapure water, dilute to 100 ml, and filter for later use;

[0041] S2. Prepare separation gel, mix acrylamide solution, buffer system, binuclear metal complex solution, divalent metal ion solution, ultrapure water and ammonium persulfate solution, add tetramethylethylenediamine just before pouring the gel, mix well, pour the gel quickly, add ultrapure water pressure glue on the upper layer of the separation gel, and remove it after solidification;

[0042] S3. Prepare concentrated gel. Mix acrylamide solution, buffer system, ultrapure water and ammonium persulfate solution. Add tetramethylethylenediamine just before pouring the gel. Mix well. Quickly pour the gel. Insert a comb into the concentrated gel and remove it after solidification.

[0043] In step S1, 28-30 g of acrylamide powder and 0.8-1.2 g of methylene bisacrylamide powder are weighed, dissolved in ultrapure water, fixed to 100 ml, and filtered through a 0.45 μm filter membrane for later use.

[0044] Specifically, using the right amount of acrylamide and methylenebisacrylamide can control the grid structure of the precast gel to ensure that it is suitable for electrophoretic separation of phosphorylated proteins. Membrane filtration can remove insoluble impurities and improve the uniformity and stability of the gel.

[0045] In step S2, 3-3.5 ml of acrylamide solution, 0.8-1.2 ml of buffer system, 150-250 μl of 5 mmol compound T67772 solution, 150-250 μl of 10 mmol ZnCl2 solution, 5-5.5 ml of ultrapure water and 60-80 μl of ammonium persulfate solution are mixed, and 4-6 μl of tetramethylethylenediamine is added before pouring the glue. The mixture is mixed evenly, and the glue is poured quickly. After pouring the glue, ultrapure water is added to press the glue, and the ultrapure water on the upper layer of the separation gel is removed after solidification.

[0046] Specifically, the uniformity and gelation speed of the separation gel are ensured through a reasonable component ratio and operation sequence. The added compound T67772 and ZnCl2 solution effectively capture phosphorylated proteins, thereby reducing their mobility and achieving good separation of phosphorylated proteins and non-phosphorylated proteins.

[0047] In step S3, 0.5-0.7 ml of acrylamide solution, 0.3-0.5 ml of buffer system, 2.8-3.0 ml of ultrapure water and 25-35 μl of 10% ammonium persulfate solution are taken, 1-3 μl of tetramethylethylenediamine is added just before pouring the glue, the mixture is mixed evenly, and the glue is poured quickly. After pouring the glue, a comb is inserted into the concentrated glue, and the concentrated glue is taken out after solidification, and stored in the buffer system solution, and refrigerated at a temperature of 2 to 8°C.

[0048] Specifically, the purpose of the concentration gel is to aggregate the samples so that the sample bands are more concentrated before entering the separation gel, thereby improving the accuracy and resolution of protein separation. Precast gels can maintain their stability for more than 12 months when stored at an appropriate temperature, facilitating long-term use in the laboratory, saving time and costs for repeated preparation, and ensuring the consistency and repeatability of experimental results.

[0049] The buffer system is tris(hydroxymethyl)aminomethane-glycine, bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane) or HEPES-tris(hydroxymethyl)aminomethane, and the pH value of the buffer system is neutral.

[0050] Specifically, selecting a suitable buffer system can ensure a stable pH environment during electrophoresis, reduce the degradation rate of the three-dimensional network structure of polyacrylamide, and be suitable for long-term storage and detection.

[0051] The following is an introduction in conjunction with specific embodiments:

[0052] Example 1: A method for preparing a precast polyacrylamide gel suitable for separation of phosphorylated proteins

[0053] S1: Preparation of 10mM ZnCl2

[0054]

[0055] Note: 10 mM ZnCl2 should be prepared and used immediately. If it is not used up at one time, please discard it and re-prepare it for next use.

[0056] S2: Preparation of 30% acrylamide solution

[0057] Weigh 29 g of acrylamide powder and 1 g of methylenebisacrylamide powder and dissolve them in ultrapure water, make up to 100 mL, and filter with a 0.45 μm filter membrane for later use.

[0058] S3: 10% separation gel preparation

[0059] Take 3.33mL of 30% acrylamide solution, 1mL of 10×Tris-glycine buffer, 200μL of 5mM compound T67772 solution, 200μL of 10 mM ZnCl2, 5.19mL of ultrapure water, and 70μL of 10% ammonium persulfate solution and mix them evenly. Add 5μL of tetramethylethylenediamine before pouring the gel, mix evenly, and pour the gel quickly. Add ultrapure water to press the gel, wait for it to solidify, and then remove the ultrapure water on the top of the separation gel.

[0060] S4: 4.5% stacking gel preparation

[0061] Take 0.6mL of 30% acrylamide solution, 0.4mL of 10×Tris-glycine buffer, 2.97mL of ultrapure water, and 28μL of 10% ammonium persulfate solution and mix them evenly. Add 2μL of tetramethylethylenediamine before pouring the gel, mix evenly, and pour the gel quickly. Add the upper layer of the separation gel and insert the comb. After solidification, take out the gel and store it in the buffer system solution, and store it in a refrigerator at 2-8℃.

[0062] Example 2: A method for preparing a polyacrylamide precast gel suitable for separation of phosphorylated proteins

[0063] S1: Preparation of 10mM ZnCl2

[0064]

[0065] Note: 10 mM ZnCl2 should be prepared and used immediately. If it is not used up at one time, please discard it and re-prepare it for next use.

[0066] S2: Preparation of 30% acrylamide solution

[0067] Weigh 29 g of acrylamide powder and 1 g of methylenebisacrylamide powder and dissolve them in ultrapure water, make up to 100 mL, and filter with a 0.45 μm filter membrane for later use.

[0068] S3: 10% separation gel preparation

[0069] Take 3.33mL of 30% acrylamide solution, 1mL of 10×Hepes-Tris buffer, 100μL of 5mM compound T67772 solution, 100μL of 10 mM ZnCl2, 5.37mL of ultrapure water, and 100μL of 10% ammonium persulfate solution and mix them evenly. Add 10μL of tetramethylethylenediamine before pouring the gel, mix evenly, and pour the gel quickly. Add ultrapure water to press the gel, wait for it to solidify, and then remove the ultrapure water on the top of the separation gel.

[0070] S4: 4.5% stacking gel preparation

[0071] Take 0.6mL of 30% acrylamide solution, 0.4mL of 10×Hepes-Tris buffer, 2.96mL of ultrapure water, and 40μL of 10% ammonium persulfate solution and mix them evenly. Add 4μL of tetramethylethylenediamine before pouring the gel, mix evenly, and pour the gel quickly. Add the upper layer of the separation gel and insert the comb. After solidification, take out the gel and store it in the buffer system solution, and store it in a refrigerator at 2-8℃.

[0072] Example 3: A method for preparing a polyacrylamide precast gel suitable for separation of phosphorylated proteins

[0073] S1: Preparation of 10mM ZnCl2

[0074]

[0075] Note: 10 mM ZnCl2 should be prepared and used immediately. If it is not used up at one time, please discard it and re-prepare it for next use.

[0076] S2: Preparation of 30% acrylamide solution

[0077] Weigh 29 g of acrylamide powder and 1 g of methylenebisacrylamide powder and dissolve them in ultrapure water, make up to 100 mL, and filter with a 0.45 μm filter membrane for later use.

[0078] S3: 10% separation gel preparation

[0079] Take 3.33mL of 30% acrylamide solution, 2.5mL of 4×Bis-Tris buffer, 100μL of 5mM compound T67772 solution, 100μL of 10 mM ZnCl2, 3.87mL of ultrapure water, and 100μL of 10% ammonium persulfate solution and mix them evenly. Add 10μL of tetramethylethylenediamine before pouring the gel, mix evenly, and pour the gel quickly. Add ultrapure water to press the gel, wait for it to solidify, and then remove the ultrapure water on the top of the separation gel.

[0080] S4: 4.5% stacking gel preparation

[0081] Take 0.6mL of 30% acrylamide solution, 1mL 4×Bis-Tris buffer, 2.36mL ultrapure water, and 40μL 10% ammonium persulfate solution and mix well. Add 4μL tetramethylethylenediamine before pouring the gel, mix well, and pour the gel quickly. Add the upper layer of the separation gel and insert the comb. After solidification, take out the gel and store it in the buffer system solution, and store it in a refrigerator at 2-8℃.

[0082] Example 4: A method for preparing a special protein molecular weight marker suitable for phosphorylated protein precast gel

[0083] Use Wansheng Haotian's three-color pre-stained molecular weight marker MKPX006, take 100μL MKPX006, add 100μL 10mM ZnCl2 solution, mix well, and store at -20℃ for later use.

[0084] Example 5: A method for preparing a special protein molecular weight marker suitable for phosphorylated protein precast gel

[0085] Use Wansheng Haotian's three-color pre-stained molecular weight marker MKPX006, take 100μL MKPX006, add 100μL 10mM MnCl2 solution, mix well, and store at -20℃ for later use.

[0086] Example 6: An electrophoretic method for separating phosphorylated proteins using precast gels

[0087] S1: Sample preparation

[0088] Ovalbumin: Weigh 10 mg (0.01 g) of ovalbumin and dissolve it in ultrapure water to make up to 10 mL.

[0089] Casein: Weigh 10 mg (0.01 g) of casein and add it to 1 mL of 100 mM Tris-HCl buffer (pH 8.0) to completely dissolve. After dissolution, add 9 mL of ultrapure water to make up to 10 mL.

[0090] Treatment with alkaline phosphatase:

[0091]

[0092] After the above system is prepared, mix gently and incubate at 37℃ for 120min. After the incubation time is over, add 30μl 3× loading buffer directly, incubate at 95℃ for 5min and then load the sample.

[0093] S2: Preparation of electrophoresis buffer: weigh 3.02 g Tris, 14.4 g glycine, and 1% SDS, adjust the pH to 8.3, and make up to 1 L.

[0094] S3: Take the precast gel prepared in Example 1 and add electrophoresis buffer for electrophoresis, load 20ul of sample into each lane, and perform electrophoresis at a constant current of 30mA / gel until bromophenol blue runs out of the gel.

[0095] Example 7: An electrophoretic method for separating phosphorylated proteins using precast gels

[0096] S1: Sample preparation

[0097] Ovalbumin: Weigh 10 mg (0.01 g) of ovalbumin and dissolve it in ultrapure water to make up to 10 mL.

[0098] Casein: Weigh 10 mg (0.01 g) of casein and add it to 1 mL of 100 mM Tris-HCl buffer (pH 8.0) to completely dissolve. After dissolution, add 9 mL of ultrapure water to make up to 10 mL.

[0099] Treatment with alkaline phosphatase:

[0100]

[0101]

[0102] After the above system is prepared, mix gently and incubate at 37℃ for 120min. After the incubation time is over, add 30μl 3× loading buffer directly, incubate at 95℃ for 5min and then load the sample.

[0103] S2: Preparation of electrophoresis buffer: HEPES electrophoresis buffer

[0104] S3: Take the precast gel prepared in Example 2 and add electrophoresis buffer for electrophoresis, load 20ul of sample into each lane, and perform electrophoresis at a constant current of 30mA / gel until bromophenol blue runs out of the gel.

[0105] Example 8: An electrophoretic method for separating phosphorylated proteins using precast gels

[0106] S1: Sample preparation

[0107] Ovalbumin: Weigh 10 mg (0.01 g) of ovalbumin and dissolve it in ultrapure water to make up to 10 mL.

[0108] Casein: Weigh 10 mg (0.01 g) of casein and add it to 1 mL of 100 mM Tris-HCl buffer (pH 8.0) to completely dissolve. After dissolution, add 9 mL of ultrapure water to make up to 10 mL.

[0109] Treatment with alkaline phosphatase:

[0110]

[0111]

[0112] After the above system is prepared, mix gently and incubate at 37℃ for 120min. After the incubation time is over, add 30μl 3× loading buffer directly, incubate at 95℃ for 5min and then load the sample.

[0113] S2: Preparation of electrophoresis buffer: ① Preparation of 0.5M sodium bisulfite: weigh 5.2g sodium bisulfite and dissolve it in 100mL water; ② 5×Bis-Tris buffer: weigh 60.6g Tris, 104.6g MOPS, and 5g SDS and dissolve them in water, and make up to 1L; ③ When using, measure 200mL 5×Bis-Tris buffer and 10mL 0.5M sodium bisulfite and make up to 1L.

[0114] S3: Take the precast gel prepared in Example 3 and add electrophoresis buffer for electrophoresis, load 20ul of sample into each lane, and perform electrophoresis at a constant current of 30mA / gel until bromophenol blue runs out of the gel.

[0115] Example 9: Stability test of a precast polyacrylamide gel suitable for separation of phosphorylated proteins

[0116] The precast gel was prepared using the method described in Example 1. After the gel solidified, it was packaged according to normal steps and stored in a refrigerator at 2-8° C. The product performance was tested according to the date until the product performance test failed.

[0117] Comparative Example 1: Electrophoresis of phosphorylated proteins using ordinary polyacrylamide gel

[0118] Gel preparation: The preparation method is different from that of Example 1 in that there is no compound T67772 and divalent metal ions in the gel.

[0119] S1: 10% separation gel preparation

[0120] Take 3.33mL of 30% acrylamide solution, 1mL of 10×Tris-glycine buffer, 5.59mL of ultrapure water, and 70μL of 10% ammonium persulfate solution and mix them evenly. Add 5μL of tetramethylethylenediamine before pouring the gel, mix evenly, and pour the gel quickly. Add ultrapure water to press the gel, wait for it to solidify, and then remove the ultrapure water on the top of the separation gel.

[0121] S2: 4.5% stacking gel preparation

[0122] Take 0.6mL of 30% acrylamide solution, 0.4mL of 10×Tris-glycine buffer, 2.97mL of ultrapure water, and 28μL of 10% ammonium persulfate solution and mix them evenly. Add 2μL of tetramethylethylenediamine before pouring the gel, mix evenly, and pour the gel quickly. Add the upper layer of the separation gel and insert the comb. After solidification, take out the gel and store it in the buffer system solution, and store it in a refrigerator at 2-8℃.

[0123] Sample preparation and electrophoresis were the same as in Example 6.

[0124] In the attached figure Figure 2The electrophoresis result of Example 6, the lanes from left to right are non-dephosphorylated ovalbumin, ovalbumin dephosphorylated by alkaline phosphatase, non-dephosphorylated casein, casein dephosphorylated by alkaline phosphatase and protein molecular weight standard.

[0125] In the attached figure Figure 3 This is the electrophoresis result of Control Example 1. The lanes from left to right are also non-dephosphorylated ovalbumin, ovalbumin dephosphorylated by alkaline phosphatase, non-dephosphorylated casein, casein dephosphorylated by alkaline phosphatase, and protein molecular weight standard.

[0126] Figure 2 and Figure 3 The comparison shows that the overall migration rate of the protein in the precast gel prepared in Example 1 of the present invention is lower than that in Control Example 1, and the electrophoresis speed is slower. Comparing the phosphorylated and dephosphorylated proteins, it is found that the dephosphorylated protein is electrophoretically located lower than the phosphorylated protein, and the migration rate is slower. In ordinary polyacrylamide gel electrophoresis, the positions of the ovalbumin in the two lanes of phosphorylated and dephosphorylated proteins are close and difficult to distinguish clearly; while the different forms of ovalbumin in the two lanes of Example 6 of the present invention are clearly separated; the same is true for casein. This proves the ability of the precast gel of the present invention to separate phosphorylated proteins from non-phosphorylated proteins.

[0127] Figure 5 This indicates that the precast gel of the present invention still has the ability to separate phosphorylated proteins from non-phosphorylated proteins after being refrigerated for 12 months, proving that the precast gel of the present invention has good stability and is suitable for long-term storage.

[0128] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A precast gel for long-term storage of phosphorylated protein separation electrophoresis, characterized in that: It comprises a polyacrylamide precast gel, wherein the polyacrylamide precast gel contains a binuclear metal complex capable of specifically binding to a phosphorylated group, wherein the binuclear metal complex is compound T67772, and divalent metal ions are added to the polyacrylamide precast gel, wherein the divalent metal ions are zinc ions or manganese ions.

2. The precast gel for long-term storage of phosphorylated protein separation electrophoresis according to claim 1, characterized in that: The concentration of the divalent metal ions in the polyacrylamide precast gel is twice that of compound T67772.

3. The precast gel for phosphorylated protein separation electrophoresis that can be stored for a long time according to claim 1, characterized in that: The concentration of the compound T67772 in the polyacrylamide precast gel is in the range of 10 to 300 micromoles.

4. The precast gel for long-term storage of phosphorylated protein separation electrophoresis according to claim 1, characterized in that: The polyacrylamide precast gel consists of an upper concentrated gel and a lower separation gel, and the compound T67772 and the divalent metal ions are added to the lower separation gel.

5. A method for preparing a precast gel for long-term storage of phosphorylated protein separation electrophoresis, characterized in that: The precast gel for long-term storage of phosphorylated protein separation electrophoresis according to any one of claims 1 to 4 comprises the following steps: S1. Prepare acrylamide solution by dissolving acrylamide powder and methylenebisacrylamide powder in ultrapure water, dilute to 100 ml, and filter for later use; S2, prepare separation gel, mix acrylamide solution, buffer system solution, binuclear metal complex solution, divalent metal ion solution, ultrapure water and ammonium persulfate solution, add tetramethylethylenediamine just before pouring the gel, mix well, pour the gel quickly, add ultrapure water pressure glue on the upper layer of the separation gel, and remove it after solidification; S3. Prepare concentrated gel. Mix acrylamide solution, buffer system solution, ultrapure water and ammonium persulfate solution. Add tetramethylethylenediamine just before pouring the gel. Mix well. Quickly pour the gel. Insert a comb into the concentrated gel and take it out after solidification to obtain precast gel. Store it in the buffer system solution at 2-8°C.

6. The method for preparing a precast gel for phosphorylated protein separation electrophoresis that can be stored for a long time according to claim 5, characterized in that: In the step S1, 28-30 g of acrylamide powder and 0.8-1.2 g of methylene bisacrylamide powder are weighed, dissolved in ultrapure water, fixed to 100 ml, and filtered through a 0.45 μm filter membrane for later use.

7. The method for preparing a precast gel for long-term storage of phosphorylated protein separation electrophoresis according to claim 5, characterized in that: In the step S2, 3-3.5 ml of acrylamide solution, 0.8-1.2 ml of buffer system, 150-250 μl of 5 mmol compound T67772 solution, 150-250 μl of 10 mmol ZnCl2 solution, 5-5.5 ml of ultrapure water and 60-80 μl of ammonium persulfate solution are mixed, and 4-6 μl of tetramethylethylenediamine is added before pouring the gel, and the mixture is mixed evenly. The gel is quickly poured to obtain a separation gel, and ultrapure water is added to press the gel after pouring the gel. After solidification, the ultrapure water on the upper layer of the separation gel is removed.

8. The method for preparing a precast gel for long-term storage of phosphorylated protein separation electrophoresis according to claim 5, characterized in that: In the step S3, 0.5-0.7 ml of acrylamide solution, 0.3-0.5 ml of buffer system, 2.8-3.0 ml of ultrapure water and 25-35 μl of 10% ammonium persulfate solution are taken, 1-3 μl of tetramethylethylenediamine is added before pouring the glue, the mixture is evenly mixed, and the glue is quickly poured to obtain concentrated glue. After pouring the glue, a comb is inserted into the concentrated glue, and it is taken out after solidification to obtain a precast glue, which is stored in a buffer system solution and refrigerated at a temperature of 2 to 8°C.

9. The method for preparing a precast gel for long-term storage of phosphorylated protein separation electrophoresis according to claim 5, characterized in that: The buffer system is tris(hydroxymethyl)aminomethane-glycine, bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane) or HEPES-tris(hydroxymethyl)aminomethane, and the pH value of the buffer system is neutral.

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