Pre-dyed protein Marker as well as preparation method and application thereof

Abstract

The invention relates to a pre-dyed protein Marker as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention provides a method for preparing a pre-dyed protein Marker, which comprises the following steps: closing a non-target amino acid in the protein Marker, or mutating the non-target amino acid in the protein Marker into a non-reactive amino acid to obtain a modified protein Marker; and mixing a dye with the modified protein Marker, and carrying out nucleophilic addition or nucleophilic substitution reaction to obtain the pre-dyed protein Marker. According to the method, the primary structure of the protein is modified, and the types and the number of modifiable group reactions are strictly controlled, so that the reaction efficiency of the protein and the band shape of the modified protein are strictly controlled, and the purpose of ensuring that the modified protein band is sharp and not dispersed is achieved.

Description

Technical Field

[0001] The invention relates to a prestained protein marker and a preparation method and application thereof, belonging to the field of biotechnology. Background Art

[0002] Protein molecular weight standards, often called protein markers, are used as a standard for protein molecular weight in protein SDS-PAGE electrophoresis and protein immunoblotting (Western-blot) experiments. Only when the marker is accurate can the experimental results be convincing. In addition, protein markers can also indicate whether the electrophoresis is normal, whether the Western-blot transfer is successful, and other information. Therefore, choosing the right protein marker is one of the necessary conditions for the success of Western-blot experiments.

[0003] Prestained protein markers are made by chemically modifying each protein in ordinary protein markers and covalently coupling them with dyes of different colors, which makes it easy for researchers to easily identify and monitor the electrophoresis and transfer of target proteins with the naked eye. Researchers can monitor the electrophoresis process throughout the protein electrophoresis, evaluate the transfer efficiency during the Western-blot transfer process, and locate the target protein. The emergence of prestained protein markers has brought great convenience to Western-blot. For example, β-ethylsulfone sulfate in vinyl sulfone type active dyes undergoes elimination reaction in alkaline medium, removes sulfate group to form vinyl sulfone group, and undergoes nucleophilic addition with protein to form covalent bond, so as to achieve the purpose of protein visualization during SDS-PAGE electrophoresis.

[0004] However, when proteins and dyes are covalently coupled, the molecular weight will also change. As the amount of dye input changes, the molecular weight of the modified protein will also change. In the nucleophilic addition reaction, the amino group, sulfhydryl group and hydroxyl group of the protein will react with the dye. The amino acid composition and structure of the protein are complex, and the reaction efficiency of the hydrophilic and hydrophobic groups and the reaction efficiency between different groups are different. The molecular weight of the protein after modification is not uniform, which will affect the band shape of the protein, and it is easy to diffuse and reduce sharpness. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for preparing a prestained protein marker, the method comprising: performing protein modification on the protein marker to obtain a modified protein marker; mixing the dye with the modified protein marker and performing a nucleophilic addition reaction or a nucleophilic substitution reaction to obtain a prestained protein marker;

[0006] The protein modification of the protein marker comprises: using a group on an amino acid that can undergo a nucleophilic addition or nucleophilic substitution reaction with a dye as a reactive group, selecting any one of the reactive groups as a target group, and the remaining reactive groups as non-target groups;

[0007] Amino acids with reactive groups are used as reactive amino acids, and amino acids without reactive groups are used as non-reactive amino acids. Among the reactive amino acids, amino acids with target groups are used as target amino acids, and amino acids with non-target groups are used as non-target amino acids.

[0008] The non-target amino acids in the protein marker are blocked, or the non-target amino acids in the protein marker are mutated into non-reactive amino acids to obtain a modified protein marker.

[0009] In one embodiment of the present invention, the mutating the non-target amino acid in the protein marker into a non-reactive amino acid is: mutating the non-target amino acid in the protein marker into a non-reactive amino acid with the same or similar properties.

[0010] In one embodiment of the present invention, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid by mutating the non-target amino acid in the hydrophilic region of the protein marker into a non-reactive amino acid with the same or similar properties.

[0011] In one embodiment of the present invention, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid by mutating the non-target amino acid in the hydrophilic region of the protein marker away from the hydrophobic region into a non-reactive amino acid with the same or similar properties.

[0012] In one embodiment of the present invention, the non-target amino acids in the protein marker are mutated into non-reactive amino acids by mutating the non-target amino acids at least 4 to 5 amino acids away from the hydrophobic region in the hydrophilic region of the protein marker into non-reactive amino acids with the same or similar properties.

[0013] In one embodiment of the present invention, the protein modification of the protein marker further comprises: mutating the target amino acid in the hydrophobic region of the protein marker into a non-target amino acid.

[0014] In one embodiment of the present invention, the protein modification of the protein marker further comprises: adjusting the positions of two adjacent target amino acids in the protein marker;

[0015] The adjustment includes: mutating one of two adjacent target amino acids in the protein marker into a non-reactive amino acid, or shifting one of two adjacent target amino acids in the protein marker to 4 to 5 non-target amino acids.

[0016] In one embodiment of the present invention, the dye comprises a vinyl sulfone type dye, an s-triazine type reactive dye, a pyrimidine type reactive dye and / or a composite dye; the composite dye comprises a composite dye of a vinyl sulfone type and an s-triazine type;

[0017] When the dye is a vinyl sulfone dye, the group capable of undergoing a nucleophilic addition reaction with the dye includes an amino group (-NH 2 ), thiol (-SH) and / or hydroxyl (-OH);

[0018] When the dye is a triazine type reactive dye, the group capable of undergoing nucleophilic substitution reaction with the chloride ion of the dye includes phenolic hydroxyl and / or amino groups (-NH 2 );

[0019] When the dye is a pyrimidine type reactive dye, the groups that can undergo nucleophilic substitution reaction with the fluoride ion of the dye include alcohol hydroxyl group (R-OH), phenolic hydroxyl group and / or amino groups (-NH 2 );

[0020] When the dye is a composite dye of vinyl sulfone type and s-triazine type, the group capable of undergoing nucleophilic addition or nucleophilic substitution reaction with the dye includes phenolic hydroxyl Amino (-NH 2 ), thiol (-SH) and / or hydroxyl (-OH).

[0021] In one embodiment of the present invention, when the dye is a vinyl sulfone dye, the reactive amino acids include cysteine ​​with a thiol group on the side chain (ionization constant pKa is 8.8-9.1), lysine with an ε-amino group on the side chain (ionization constant pKa is 9.3-9.5), histidine with an imidazole group on the side chain (ionization constant pKa is 6.7-7.1), arginine with a guanidino group on the side chain (ionization constant pKa>12), N-terminal amino acid with an α-amino group on the side chain (ionization constant pKa is 7.6-8.0), C-terminal amino acid with an α-hydroxyl group on the side chain (ionization constant pKa is 2.1-2.4), Aspartic acid with a γ-hydroxyl group (ionization constant pKa is 3.7-4.0), glutamic acid with a β-hydroxyl group on the side chain (ionization constant pKa is 4.2-4.5) and / or tyrosine with a phenolic hydroxyl group on the side chain (ionization constant pKa is 9.7-10.1), the non-reactive amino acids include leucine, isoleucine, asparagine, glutamine, methionine, phenylalanine and / or valine (when sulfhydryl and hydroxyl groups are selected as reactive groups, arginine is the reactive amino acid, but since the ionization constant pKa of the amino group on the side chain of arginine is greater than 12, when the amino group is selected as the reactive group, arginine is additionally added as the non-reactive amino acid);

[0022] When the dye is a s-triazine type reactive dye, the target amino acid includes tyrosine, arginine, N-terminal amino acid and / or histidine, and the non-reactive amino acid includes asparagine, aspartic acid, glutamine, cysteine ​​and / or methionine;

[0023] When the dye is a pyrimidine-type reactive dye, the target amino acid includes an N-terminal amino acid, a C-terminal amino acid, aspartic acid, and / or lysine, and the non-reactive amino acid includes cysteine, methionine, asparagine and / or glutamine.

[0024] In one embodiment of the present invention, when the dye is a vinylsulfone dye and an amino group is selected as the reactive group, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid with the same or similar properties, including: mutating cysteine ​​with a sulfhydryl group on the side chain of the protein marker into methionine, mutating aspartic acid with a γ-hydroxyl group on the side chain of the protein marker into asparagine, mutating glutamic acid with a β-hydroxyl group on the side chain of the protein marker into glutamine, and / or mutating tyrosine with a phenolic hydroxyl group on the side chain of the protein marker into phenylalanine;

[0025] When the dye is a vinylsulfone dye and a thiol group is selected as the reactive group, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid with the same or similar properties, including: mutating a lysine with an ε-amino group on the side chain of the protein marker into arginine, valine and / or isoleucine, mutating an aspartic acid with a γ-hydroxyl group on the side chain of the protein marker into asparagine, mutating a glutamic acid with a β-hydroxyl group on the side chain of the protein marker into glutamine, and / or mutating a tyrosine with a phenolic hydroxyl group on the side chain of the protein marker into phenylalanine;

[0026] When the dye is a vinylsulfone dye and a hydroxyl group is selected as the reactive group, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid with the same or similar properties, including: mutating a cysteine ​​with a sulfhydryl group on the side chain of the protein marker into a methionine, and / or mutating a lysine with an ε-amino group on the side chain of the protein marker into an arginine, a valine and / or an isoleucine.

[0027] In one embodiment of the present invention, when the dye is a vinyl sulfone type dye and an amino group is selected as the reactive group, the nucleophilic addition reaction is carried out under the condition of pH ≥ 9.5;

[0028] When the dye is a vinyl sulfone dye and a thiol group is selected as the reactive group, the nucleophilic addition reaction is carried out under the condition of 9.5>pH≥8.5;

[0029] When the dye is a vinyl sulfone dye and a hydroxyl group is selected as the reactive group, the nucleophilic addition reaction is carried out under the condition of 8.5>pH≥8.0.

[0030] In one embodiment of the present invention, the nucleophilic addition reaction includes: dissolving the dye in a buffer solution containing sodium dodecyl sulfate (SDS) to obtain a dissolving solution; replacing the modified protein marker into a buffer solution containing sodium dodecyl sulfate (SDS) and concentrating the solution to obtain a concentrated solution; adding a reducing agent to the concentrated solution, incubating at 70°C for 15 to 30 minutes to obtain an incubation solution; mixing the incubation solution and the dissolving solution, incubating at 50 to 70°C for 2 to 5 hours to obtain a reaction solution containing a prestained protein marker; extracting the reaction solution to obtain a prestained protein marker.

[0031] In one embodiment of the present invention, the concentration of the dye in the dissolving solution is 30-80 mg / mL; the concentration of the modified protein Marker in the concentrated solution is 3-5 mg / mL; the amount of the reducing agent added in the concentrated solution is 0.2-0.6% (g / 100 mL) in terms of mass volume percentage; the concentration of the sodium dodecyl sulfate in the buffer solution is 1-3% (g / 100 mL) in terms of mass volume percentage.

[0032] In one embodiment of the present invention, the mixing volume ratio of the incubation solution and the dissolving solution is 1:(0.5-1).

[0033] In one embodiment of the present invention, the reducing agent includes tributylphosphine, TECP (tri(2-carbonylethyl)phosphine hydrochloride), DTT (dithiothreitol) and / or β-mercaptoethanol; the buffer includes carbonate buffer, phosphate buffer and / or nitrate buffer.

[0034] The present invention also provides a prestained protein Marker, which is prepared by the above method.

[0035] The present invention also provides a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis method, which is not for the purpose of disease diagnosis and treatment, and uses the above-mentioned prestained protein Marker.

[0036] The present invention also provides a protein immunoblotting (Western-blot) experimental method, which is not for the purpose of disease diagnosis and treatment, and uses the above-mentioned prestained protein Marker.

[0037] The present invention also provides a protein modification method for preparing a pre-stained protein marker, the protein modification method comprising: using a group on an amino acid that can undergo a nucleophilic addition or nucleophilic substitution reaction with a dye as a reactive group, selecting any one of the reactive groups as a target group, and the remaining reactive groups as non-target groups;

[0038] Amino acids with reactive groups are used as reactive amino acids, and amino acids without reactive groups are used as non-reactive amino acids. Among the reactive amino acids, amino acids with target groups are used as target amino acids, and amino acids with non-target groups are used as non-target amino acids.

[0039] The non-target amino acids in the protein marker are blocked, or the non-target amino acids in the protein marker are mutated into non-reactive amino acids to obtain a modified protein marker.

[0040] In one embodiment of the present invention, the mutating the non-target amino acid in the protein marker into a non-reactive amino acid is: mutating the non-target amino acid in the protein marker into a non-reactive amino acid with the same or similar properties.

[0041] In one embodiment of the present invention, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid by mutating the non-target amino acid in the hydrophilic region of the protein marker into a non-reactive amino acid with the same or similar properties.

[0042] In one embodiment of the present invention, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid by mutating the non-target amino acid in the hydrophilic region of the protein marker away from the hydrophobic region into a non-reactive amino acid with the same or similar properties.

[0043] In one embodiment of the present invention, the non-target amino acids in the protein marker are mutated into non-reactive amino acids by mutating the non-target amino acids at least 4 to 5 amino acids away from the hydrophobic region in the hydrophilic region of the protein marker into non-reactive amino acids with the same or similar properties.

[0044] In one embodiment of the present invention, the protein modification method further comprises: mutating the target amino acid in the hydrophobic region of the protein marker into a non-target amino acid.

[0045] In one embodiment of the present invention, the protein modification method further comprises: adjusting the positions of two adjacent target amino acids in the protein marker;

[0046] The adjustment includes: mutating one of two adjacent target amino acids in the protein marker into a non-reactive amino acid, or shifting one of two adjacent target amino acids in the protein marker to 4 to 5 non-target amino acids.

[0047] In one embodiment of the present invention, the dye comprises a vinyl sulfone type dye, an s-triazine type reactive dye, a pyrimidine type reactive dye and / or a composite dye; the composite dye comprises a composite dye of a vinyl sulfone type and an s-triazine type;

[0048] When the dye is a vinyl sulfone dye, the group capable of undergoing a nucleophilic addition reaction with the dye includes an amino group (-NH 2 ), thiol (-SH) and / or hydroxyl (-OH);

[0049] When the dye is a triazine type reactive dye, the group capable of undergoing nucleophilic substitution reaction with the chloride ion of the dye includes phenolic hydroxyl and / or amino groups (-NH 2 );

[0050] When the dye is a pyrimidine type reactive dye, the groups that can undergo nucleophilic substitution reaction with the fluoride ion of the dye include alcohol hydroxyl group (R-OH), phenolic hydroxyl group and / or amino groups (-NH2 );

[0051] When the dye is a composite dye of vinyl sulfone type and s-triazine type, the group capable of undergoing nucleophilic addition or nucleophilic substitution reaction with the dye includes phenolic hydroxyl Amino (-NH 2 ), thiol (-SH) and / or hydroxyl (-OH).

[0052] In one embodiment of the present invention, when the dye is a vinyl sulfone dye, the reactive amino acids include cysteine ​​with a thiol group on the side chain (ionization constant pKa is 8.8-9.1), lysine with an ε-amino group on the side chain (ionization constant pKa is 9.3-9.5), histidine with an imidazole group on the side chain (ionization constant pKa is 6.7-7.1), arginine with a guanidino group on the side chain (ionization constant pKa>12), N-terminal amino acid with an α-amino group on the side chain (ionization constant pKa is 7.6-8.0), C-terminal amino acid with an α-hydroxyl group on the side chain (ionization constant pKa is 2.1-2.4), Aspartic acid with a γ-hydroxyl group (ionization constant pKa is 3.7-4.0), glutamic acid with a β-hydroxyl group on the side chain (ionization constant pKa is 4.2-4.5) and / or tyrosine with a phenolic hydroxyl group on the side chain (ionization constant pKa is 9.7-10.1), the non-reactive amino acids include leucine, isoleucine, asparagine, glutamine, methionine, phenylalanine and / or valine (when sulfhydryl and hydroxyl groups are selected as reactive groups, arginine is the reactive amino acid, but since the ionization constant pKa of the amino group on the side chain of arginine is greater than 12, when the amino group is selected as the reactive group, arginine is additionally added as the non-reactive amino acid);

[0053] When the dye is a s-triazine type reactive dye, the target amino acid includes tyrosine, arginine, N-terminal amino acid and / or histidine, and the non-reactive amino acid includes asparagine, aspartic acid, glutamine, cysteine ​​and / or methionine;

[0054] When the dye is a pyrimidine-type reactive dye, the target amino acid includes an N-terminal amino acid, a C-terminal amino acid, aspartic acid, and / or lysine, and the non-reactive amino acid includes cysteine, methionine, asparagine and / or glutamine.

[0055] In one embodiment of the present invention, when the dye is a vinylsulfone dye and an amino group is selected as the reactive group, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid with the same or similar properties, including: mutating cysteine ​​with a sulfhydryl group on the side chain of the protein marker into methionine, mutating aspartic acid with a γ-hydroxyl group on the side chain of the protein marker into asparagine, mutating glutamic acid with a β-hydroxyl group on the side chain of the protein marker into glutamine, and / or mutating tyrosine with a phenolic hydroxyl group on the side chain of the protein marker into phenylalanine;

[0056] When the dye is a vinylsulfone dye and a thiol group is selected as the reactive group, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid with the same or similar properties, including: mutating a lysine with an ε-amino group on the side chain of the protein marker into arginine, valine and / or isoleucine, mutating an aspartic acid with a γ-hydroxyl group on the side chain of the protein marker into asparagine, mutating a glutamic acid with a β-hydroxyl group on the side chain of the protein marker into glutamine, and / or mutating a tyrosine with a phenolic hydroxyl group on the side chain of the protein marker into phenylalanine;

[0057] When the dye is a vinylsulfone dye and a hydroxyl group is selected as the reactive group, the non-target amino acid in the protein marker is mutated into a non-reactive amino acid with the same or similar properties, including: mutating a cysteine ​​with a sulfhydryl group on the side chain of the protein marker into a methionine, and / or mutating a lysine with an ε-amino group on the side chain of the protein marker into an arginine, a valine and / or an isoleucine.

[0058] The present invention also provides a modified protein for preparing a prestained protein marker, wherein the modified protein is modified by the above protein modification method.

[0059] The present invention also provides a nucleic acid molecule for preparing a prestained protein marker, wherein the nucleic acid molecule encodes the modified protein.

[0060] The present invention also provides a recombinant plasmid for preparing a prestained protein marker, wherein the recombinant plasmid expresses the modified protein.

[0061] In one embodiment of the present invention, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a phage vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a hybrid virus vector, a baculovirus vector, a herpes simplex virus vector or a lentivirus vector; the non-viral vector includes a plasmid vector.

[0062] In one embodiment of the present invention, the plasmid vector comprises pET28a plasmid, pET32a plasmid, pBAD-hisA plasmid, pQE30 plasmid and / or pPIC9K plasmid.

[0063] In one embodiment of the present invention, the method for preparing the recombinant plasmid is: inserting the above nucleic acid molecule into a vector to obtain the recombinant plasmid.

[0064] The present invention also provides a host cell for preparing a prestained protein marker, wherein the host cell expresses the modified protein.

[0065] In one embodiment of the present invention, the host cell comprises fungi, bacteria, plant cells and / or animal cells.

[0066] In one embodiment of the present invention, the bacteria include Escherichia coli and / or Pichia pastoris.

[0067] In one embodiment of the present invention, the Escherichia coli includes Escherichia coli BL21 (DE3), Escherichia coli ER2566, and Escherichia coli Rosetta (DE3); the Pichia pastoris includes Pichia pastoris GS115.

[0068] In one embodiment of the present invention, the host cell is prepared by transforming the above-mentioned recombinant plasmid into the host cell.

[0069] The present invention also provides the use of the above method or the above prestained protein marker or the above protein modification method or the above modified protein or the above nucleic acid molecule or the above recombinant plasmid or the above host cell in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis or protein immunoblotting (Western-blot) experiments, and the application is not for the purpose of disease diagnosis and treatment.

[0070] The technical solution of the present invention has the following advantages:

[0071] The present invention provides a method for preparing a pre-stained protein marker, the method comprising: performing protein modification on the protein marker to obtain a modified protein marker; mixing the dye with the modified protein marker and performing nucleophilic addition or nucleophilic substitution reaction to obtain the pre-stained protein marker; the protein modification of the protein marker comprises: using a group on an amino acid that can undergo nucleophilic addition or nucleophilic substitution reaction with the dye as a reactive group, selecting any one of the reactive groups as a target group, and the remaining reactive groups as non-target groups; using an amino acid with a reactive group as a reactive amino acid, an amino acid without a reactive group as a non-reactive amino acid, an amino acid with a target group as a target amino acid, and an amino acid with a non-target group as a non-target amino acid; sealing the non-target amino acid in the protein marker, or mutating the non-target amino acid in the protein marker into a non-reactive amino acid to obtain a modified protein marker. The method strictly controls the type and quantity of the modifiable group reaction by modifying the primary structure of the protein, and then strictly controls the reaction efficiency of the protein and the band shape of the modified protein, so as to achieve the purpose of ensuring that the modified protein band is sharp and non-diffuse. In addition, the method can quickly and accurately obtain target molecular weight proteins, making the production of prestained protein marker products simpler and easier to operate.

[0072] Furthermore, the non-target amino acids in the protein marker are mutated into non-reactive amino acids by mutating the non-target amino acids at least 4 to 5 amino acids away from the hydrophobic region in the hydrophilic region of the protein marker into non-reactive amino acids with the same or similar properties. This condition can further ensure that the protein bands after modification are sharp and non-diffuse.

[0073] Furthermore, the protein modification method further comprises: mutating the target amino acid in the hydrophobic region of the protein marker into a non-target amino acid. This condition can further ensure that the protein band after modification is sharp and non-diffuse.

[0074] Furthermore, the protein modification method further includes: adjusting the positions of two adjacent target amino acids in the protein marker; the adjustment includes: mutating one of the two adjacent target amino acids in the protein marker to a non-reactive amino acid, or, shifting one of the two adjacent target amino acids in the protein marker to 4 to 5 non-target amino acids. This condition can further ensure that the protein bands after modification are sharp and non-diffuse. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 : Effect of pH on the efficiency of nucleophilic reactions.

[0076] Figure 2:Effect of dye input amount on nucleophilic reaction efficiency.

[0077] Figure 3 :The influence of thiol blocking on protein bands.

[0078] Figure 4 : Sequence comparison results of WT-10kDa before and after transformation.

[0079] Figure 5 : WT-10kDa modification detection results.

[0080] Figure 6 :MUT-10kDa modification detection results.

[0081] Figure 7 : Sequence comparison results of WT-15kDa before and after transformation.

[0082] Figure 8 : WT-15kDa modification detection results.

[0083] Fig. 9 : MUT-15kDa modification detection results.

[0084] Fig.10 : Sequence comparison results of 25kDa-WT before and after transformation.

[0085] Fig.11 :25kDa-WT and 25kDa-MUT modification detection results.

[0086] Fig.12 :Marker mixed SDS-PAGE and Western Bolt detection results. DETAILED DESCRIPTION

[0087] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0088] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0089] Experimental Example 1: Exploration of the Preparation Conditions of Prestained Protein Markers

[0090] The experimental process is as follows:

[0091] Experiment 1: Effect of pH on the efficiency of nucleophilic reactions

[0092] The β-ethylsulfone sulfate in the vinyl sulfone molecule undergoes an elimination reaction under alkaline conditions, removing the sulfate group and converting it into a vinyl sulfone group, which then undergoes a nucleophilic reaction with the protein. The groups that react with vinyl sulfone dyes include amino groups (-NH 2 ), thiol (-SH) and hydroxyl (-OH), the types of amino acids with these three groups on the side chains and their ionization constants pKa are shown in Table 1.

[0093] The strength of nucleophilic reaction between different groups and vinyl sulfone dyes: R-SH≈R-NH 2 >R2-NH≈R-OH. Select the target amino acid based on the group reaction strength, and select the optimal pH of the reaction conditions based on the amino acid side chain group dissociation constant pKa. The specific operation process is as follows:

[0094] Take the protein marker with amino acid sequence as shown in SEQ ID NO.1 as an example:

[0095] The protein marker with an amino acid sequence as shown in SEQ ID NO.1 was reverse transcribed to obtain a nucleic acid sequence encoding the protein marker (SEQ ID NO.2); the target gene was synthesized according to the nucleic acid sequence, and the target gene was inserted into the NcoI-XbaI site of the pET28a plasmid to obtain a recombinant plasmid pET28a-Marker (synthesized and constructed into a recombinant plasmid by Qingke Biotechnology Co., Ltd.).

[0096] The recombinant plasmid pET28a-Marker was transformed into competent cells of Escherichia coli BL21 (DE3) to obtain a transformation product; the transformation product was spread on an LB plate containing 50 μg / mL kanamycin (purchased from Shanghai Biotechnology) (LB plate formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 15 g / L agar, the culture medium was prepared according to the formula, sterilized at 115°C for 30 min, after the culture medium was cooled to 50°C, kanamycin was added to a final concentration of 50 μg / mL under a sterile environment, mixed and inverted, each plate had 25 mL of culture medium), incubated at 37°C for 16 hours, and then a single colony was picked for sequencing. If the sequencing was correct, the recombinant Escherichia coli BL21 (DE3) / pET28a-Marker was obtained.

[0097] A single colony of the recombinant Escherichia coli BL21 (DE3) / pET28a-Marker was picked into 20 mL of LB medium containing 50 μg / mL kanamycin (LB medium formula: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride), and cultured at 37 ° C, 220 rpm for 16 hours to obtain a bacterial solution; the bacterial solution was inoculated into 1L of LB medium containing 50 μg / mL kanamycin at an inoculum of 1% (v / v), and cultured at 37 ° C, 220 rpm until OD600 = 0.6, and then IPTG (purchased from Sigma-Aldrich) with a final concentration of 0.5 mM was added, and induced culture was carried out at 37 ° C, 120 rpm for 4 hours to obtain a culture solution; the culture solution was centrifuged to collect the bacteria; it was detected that the protein Marker was expressed in a soluble manner, so the protein Marker in the collected bacteria was purified by nickel ion affinity chromatography;

[0098] The nickel ion affinity chromatography purification process is as follows:

[0099] After the cells were fully suspended in 50 mL of lysis buffer, they were ultrasonically disrupted for 30 min (5 s of disruption, 10 s interval), centrifuged at 10,000 × g for 40 min, and the supernatant was filtered through a 0.22 μm membrane and subjected to nickel ion affinity chromatography;

[0100] The lysis buffer composition includes: 20 mM Tris-HCl, 250 mM NaCl, 0.5% (v / v) Tween-20 and 5% (w / v, g / 100 mL) glycerol, pH 8.0@25°C;

[0101] The equilibration buffer used for nickel ion affinity chromatography consisted of 20 mM Tris-HCl, 250 mM NaCl, 5 mM imidazole, and 5% (v / v) glycerol, pH 8.0@25°C;

[0102] The elution buffer used for nickel ion affinity chromatography consisted of 20 mM Tris-HCl, 250 mM NaCl, 500 mM imidazole, and 5% (v / v) glycerol, pH 8.0@25°C;

[0103] During the nickel ion affinity chromatography, 60 mM imidazole was used for washing, and 250 mM imidazole was used for eluting the target protein, which was then collected for later use.

[0104] The purified protein marker was replaced by dialysis into sodium carbonate-sodium bicarbonate buffers of different pH values ​​to obtain replacement solutions with pH values ​​of 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, and 10.5, respectively; the replacement solutions with different pH values ​​were concentrated to a protein concentration of 3.5 mg / mL using an ultrafiltration tube to obtain protein concentrates with different pH values; Remazol vinyl sulfone dye (purchased from Xingtai Ouyang Chemical Co., Ltd.) was dissolved in sodium carbonate-sodium bicarbonate buffers with pH values ​​of 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, and 10.5, respectively, to a dye concentration of 50 mg / mL. Afterwards, the mixture was filtered using a 0.22 μm membrane to obtain dye solution solutions of different pH values; the reducing agent tributylphosphine was added to the protein concentrate solutions of different pH values ​​until the reducing agent concentration was 0.5% (w / v, g / 100 mL), and the mixture was incubated at 70° C. for 20 min to obtain incubation solutions of different pH values; the incubation solutions of different pH values ​​were mixed with dye solution solutions of different pH values ​​in a ratio of incubation solution (here, the incubation solution and the dye solution correspond to each other according to the pH value): dye solution = 1:0.75 (v / v), and the mixture was incubated at 60° C. for 3 h to obtain reaction solutions containing prestained protein markers obtained by reactions under different pH conditions;

[0105] The sodium carbonate-sodium bicarbonate buffer comprises the following components: 100 mM sodium carbonate, 50 mM sodium bicarbonate and 2% (w / v, g / 100 mL) SDS (sodium dodecyl sulfate).

[0106] The reaction solution containing prestained protein marker obtained under different pH conditions was subjected to SDS-PAGE detection. The results are as follows: Figure 1 As shown, when pH ≤ 7.5, the molecular weight of the band after protein and dye modification is small; when pH = 8.5, the molecular weight of the band after the reaction of protein and dye becomes larger; after pH ≥ 9.5, the molecular weight after the reaction of protein and dye changes again; this shows that the number and type of modification groups have changed at pH 7.5, 8.5 and 9.5, respectively. According to the dissociation constant pKa of the side chain group, it is speculated that when pH ≤ 7.5, the elimination reaction of vinyl sulfone dye is incomplete, and the groups participating in the addition reaction are mainly hydroxyl groups; when pH = 8.5, the thiol group is completely ionized and participates in the reaction, and the N-terminal amino group also participates in the reaction; when pH ≥ 9.5, the side chain amino group is ionized and participates in the reaction.

[0107] Table 1 Types of amino acids with amino, thiol and hydroxyl groups on the side chains and their ionization constants pKa

[0108]

[0109]

[0110] Experiment 2: Effect of dye input on nucleophilic reaction efficiency

[0111] The amount of dye input affects the final modification effect of the protein. When the dye input is insufficient, the side chain groups of the target amino acid cannot be fully modified, and the competition between the groups will cause the reaction of some groups to be inhibited. In order to ensure that the target amino acid is fully modified, the optimal amount of dye input in the reaction system is explored. The specific operation process is as follows:

[0112] Take the protein marker with amino acid sequence as shown in SEQ ID NO.1 as an example:

[0113] according to Figure 1 The results show that the protein band effect is best when the pH is 9.5. Therefore, the purified protein marker prepared in Experiment 1 was replaced by dialysis to pH 9.5. 9.5 sodium carbonate-sodium bicarbonate buffer to obtain a replacement solution; use an ultrafiltration tube to concentrate the replacement solution to a protein concentration of 3.5 mg / mL to obtain a protein concentrate; dissolve Remazol vinyl sulfone dye in sodium carbonate-sodium bicarbonate buffer at pH 9.5 to dye concentrations of 10 mg / mL, 20 mg / mL, 30 mg / mL, and 40 mg / mL, respectively, and filter using a 0.22 μm membrane to obtain dye solutions of different concentrations; add reducing agent tributyl phosphine to the protein concentrate until the reducing agent concentration is 0.5% (w / v, g / 100 mL), incubate at 70° C. for 20 min to obtain an incubation solution; mix the incubation solution with dye solutions of different concentrations in a ratio of incubation solution: dye solution = 1:0.75 (v / v), and incubate at 60° C. for 3 h to obtain reaction solutions containing pre-stained protein markers obtained by reactions with different dye input amounts.

[0114] The reaction solution containing prestained protein marker obtained by reaction with different dye input amounts was subjected to SDS-PAGE detection. The results are as follows Figure 2 As shown, with the increase of dye input, the coupling reaction efficiency of protein and dye is higher, but too many factors affecting the reaction are not conducive to the stable preparation of pre-stained protein markers. Therefore, avoiding the difference in intermolecular reaction efficiency is crucial for the preparation of pre-stained protein markers.

[0115] Experiment 3: Effect of thiol blocking on protein bands

[0116] There are too many groups and corresponding amino acids in the protein that can react with the dye, and the reaction strength of each group is inconsistent, which can easily lead to inconsistent final amino acids that react with the dye between protein molecules. The molecular weight of the dye is between 0.6 and 1 kDa. The difference in the number of amino acids that react between molecules leads to differences in the molecular weight of a single molecule after the reaction, which is manifested in the phenomenon of unclear protein bands, low clarity, and even diffuse tailing on SDS-PAGE electrophoresis. In order to achieve the purpose of ensuring that the protein bands after modification are sharp and non-diffuse, in this experiment, a group is selected as the target group, the amino acid with the corresponding group is the target amino acid, and the other amino acids that can participate in the reaction are non-target amino acids. The reaction is called a side reaction. The non-target amino acid is blocked or mutated into an amino acid with the same or similar properties but does not participate in the reaction (non-reactive amino acid). While reducing the side reaction, the number of reactive amino acids is also reduced, ensuring the consistency and stability of the reaction efficiency between protein molecules. The specific modification plan is as follows:

[0117] Since the dye has only two hydrophilic groups, the hydrophobic supramolecular forces between molecules are obvious and the hydrophobicity is strong. When the dye undergoes a nucleophilic reaction with the protein, the β-ethylsulfone sulfate in the molecule is desulfurized under alkaline conditions and converted into a vinylsulfone group, thereby reducing the water-soluble groups, causing the solubility of the dye to drop instantly and causing the dye to aggregate. Therefore, in order to ensure the full reaction between the protein and the dye, the target amino acid in the hydrophobic region is mutated into a non-target amino acid, and at the same time, the non-target amino acid in the hydrophilic region is mutated into a target amino acid, so that the amino acid with the target group is as far away from the hydrophobic region as possible (for example, the amino acid with the target group is four to five amino acids away from the hydrophobic region).

[0118] When the protein and the dye undergo a nucleophilic reaction, under the action of the reducing agent, the disulfide bond of the protein is broken, the spatial structure is destroyed, the protein is reduced to a single peptide chain, and the steric hindrance effect is reduced. However, during the nucleophilic reaction modification process, the two adjacent target amino acids in the protein will still produce a large steric hindrance effect due to the ionization of the side chain reaction group during the reaction, affecting the reaction efficiency. Therefore, in order to further ensure the full reaction of the protein and the dye, the positions of the two adjacent target amino acids in the protein are adjusted, for example, one of the target amino acids is mutated (mutated to a non-reactive amino acid) or shifted to four to five non-target amino acids.

[0119] Based on the above modification scheme, taking the protein marker with amino acid sequence as shown in SEQ ID NO.1 as an example, the amino group is selected as the target group, and the nucleophilic reaction pH is ≥9.5. At this time, the phenolic hydroxyl group of tyrosine and the sulfhydryl group of cysteine ​​will also participate in the reaction. According to the group activity: R-SH>>phenolic hydroxyl group, the side reaction of phenolic hydroxyl group can be ignored at this time. In order to reduce the occurrence of side reactions, first try to alkylate cysteine ​​to completely block the non-target reaction group R-SH to observe the effect of this operation on the protein band. The specific operation is as follows:

[0120] The purified protein marker obtained in Experiment 1 was replaced by dialysis into a sodium carbonate-sodium bicarbonate buffer solution at pH 8.0 to obtain a replacement solution; the replacement solution was concentrated to a protein concentration of 3.5 mg / mL using an ultrafiltration tube to obtain a protein concentrate; 0.37 g of iodoacetamide was weighed and fully dissolved in 10 mL of ethanol to obtain an iodoacetamide IAM solution; IAM solution was added to the protein concentrate at a ratio of IAM solution: protein concentrate = 1:50 (v / v), and after being fully mixed, the mixture was incubated at room temperature (25°C) in the dark for 30 minutes to block the R-SH of the protein to obtain a blocked protein concentrate; Remazol vinyl sulfone dye was dissolved in pH 8.0 10.0% sodium carbonate-sodium bicarbonate buffer to a dye concentration of 50 mg / mL, filtered using a 0.22 μm membrane to obtain a dye solution; adding a reducing agent tributylphosphine to the blocked protein concentrate to a reducing agent concentration of 0.5% (w / v, g / 100 mL), incubated at 70° C. for 20 min to obtain an incubation solution; the incubation solution and the dye solution were mixed at a ratio of incubation solution: dye solution = 1:0.75 (v / v), and incubated at 60° C. for 3 h to obtain a reaction solution containing a prestained protein marker.

[0121] After the reaction solution containing the prestained protein marker returns to room temperature, it is first extracted with methanol precooled to 4°C (volume ratio of methanol: reaction solution = 4:1), then precipitated at low temperature (-20°C) for 10 minutes, and then centrifuged at 8000×g for 10 minutes, and the supernatant is discarded and the precipitate is retained; the precipitate is rinsed three times with 80% (v / v) methanol, and then redissolved with a dissolution buffer to obtain a resolution solution; the resolution solution is dialyzed overnight (16 hours) with a dialysis bag to obtain the prestained protein marker;

[0122] The dissolution buffer comprises: 62.5 mM Tris-phosphate (tris(hydroxymethyl)aminomethane phosphate (pH 7.5), 1 mM EDTA (ethylenediaminetetraacetic acid), 2% (w / v, g / 100 mL) SDS, 2 mM DTT (dithiothreitol) and 5% (v / v) glycerol;

[0123] Dialysate composition: 62.5 mM Tris-phosphate (pH 7.5), 1 mM EDTA, 2% (w / v, g / 100 mL) SDS, 2 mM DTT (dithiothreitol) and 5% (v / v) glycerol.

[0124] Take the reaction solution containing prestained protein marker for SDS-PAGE detection. The results are as follows Figure 3 As shown, after thiol blocking, the sharpness of the protein band increased. This result preliminarily confirmed the effectiveness of the modification of the reactive groups in the primary structure of the protein.

[0125] based on Figure 3 In order to further reduce the occurrence of side reactions, the pre-stained protein marker was modified according to the modification scheme to observe the effect of this operation on the protein band. The specific operation is as follows:

[0126] Take the protein marker with amino acid sequence as shown in SEQ ID NO.3 as an example:

[0127] The protein marker (abbreviated as 10kDa-WT) with an amino acid sequence as shown in SEQ ID NO.3 was reverse transcribed to obtain a nucleic acid sequence encoding the protein marker (SEQ ID NO.4).

[0128] In the protein marker (referred to as 10kDa-WT) with the amino acid sequence shown in SEQ ID NO.3, the hydroxyl group was selected as the target reactive group. The groups that can participate in the reaction in the amino acid sequence of this protein are N-terminal amino group, C-terminal hydroxyl group, aspartic acid and glutamic acid. In order to avoid the occurrence of side reactions, the highly reactive lysine and cysteine ​​in the protein were mutated into amino acids that do not participate in the reaction: C 15 Cysteine ​​mutates to methionine, i.e. C 15 M; K 29 Lysine mutates to arginine R, i.e. K 29 R; The point dissociation constant pKa of the side chain amino group of arginine is > 12. The purpose of this modification is to inhibit its reaction by controlling pH. 26 and D 29 Being in a highly hydrophobic region is not conducive to the occurrence of the reaction. To ensure the clarity and sharpness of the protein after modification, the target amino acid in the hydrophobic region is mutated to a non-target amino acid: 26 Mutation to Q, D 29 Mutation to N, ie E 26 Q and D 29N, the purpose of this modification is to ensure that the target amino acid is away from the hydrophobic region and to avoid the occurrence of strong side reactions. There is no target amino acid adjacent to the sequence of this protein marker, so the steric effect is not considered. The amino acid sequence of the modified protein marker (abbreviated as 10kDa-MUT) is shown in SEQ ID NO.5. The sequence alignment results of the unmodified protein marker (abbreviated as 10kDa-WT) and the modified protein marker (abbreviated as 10kDa-MUT) are shown in Figure 4 .

[0129] The protein marker (referred to as 10kDa-MUT) obtained by the modification of the amino acid sequence as shown in SEQ ID NO.5 was reverse transcribed to obtain a nucleic acid sequence encoding the protein marker (SEQ ID NO.6); the target gene was synthesized according to the nucleic acid sequence, and purified 10kDa-WT and 10kDa-MUT were prepared according to the method of Experiment 1; the purified 10kDa-WT and 10kDa-MUT were respectively replaced by dialysis into a sodium carbonate-sodium bicarbonate buffer at pH 8.0 to obtain a 10kDa-WT replacement solution and a 10kDa-MUT replacement solution; the replacement solution was concentrated to a protein concentration of 3.5 mg / mL using an ultrafiltration tube to obtain a 10kDa-WT protein concentrate and a 10kDa-MUT protein concentrate; Remazol vinyl sulfone dye was dissolved in a sodium carbonate-sodium bicarbonate buffer at pH 8.0 to a dye concentration of 50 mg / mL, and then filtered using a 0.22 μm membrane to obtain a dye solution; 10kDa-WT The reducing agent tributylphosphine was added to the protein concentrate and the 10kDa-MUT protein concentrate respectively until the concentration of the reducing agent was 0.5% (w / v, g / 100mL), and the mixture was incubated at 70°C for 20min to obtain 10kDa-WT incubation solution and 10kDa-MUT incubation solution; the 10kDa-WT incubation solution and the 10kDa-MUT incubation solution were respectively mixed with the dye dissolving solution at a ratio of incubation solution: dye dissolving solution = 1:0.75 (v / v), and the mixture was incubated at 60°C for 3h to obtain a reaction solution containing the prestained protein Marker 10kDa-WT and a reaction solution containing the prestained protein Marker 10kDa-MUT.

[0130] Take the prestained protein Marker 10kDa-WT and prestained protein Marker 10kDa-MUT for SDS-PAGE to detect the effect of the 10kDa-WT band and the prestained protein Marker 10kDa-MUT conditions in electrophoresis. The results are as follows: Figure 5-6As shown in the figure, after 10kDa-WT was modified by dye coupling, the effect was poor, and the phenomenon on SDS-PAGE electrophoresis was that the protein bands were not sharp, had low clarity, and even diffuse tailing. After 10kDa-MUT was modified by dye coupling, the bands were sharp and bright in color. It can be seen that the modification of the primary structure reaction groups of the protein, the mutation of the highly reactive non-target amino acids, the reaction efficiency of the protein was guaranteed at the optimal pH and dye input, and the purpose of controlling the band shape of the modified protein was achieved, so that the target molecular weight protein can be obtained quickly and accurately.

[0131] Experimental Example 2: Verification of the Preparation Conditions of Prestained Protein Markers

[0132] The experimental process is as follows:

[0133] 1. Take the protein marker with amino acid sequence as shown in SEQ ID NO.7 as an example

[0134] The protein marker (abbreviated as 15kDa-WT) with an amino acid sequence as shown in SEQ ID NO.7 was reverse transcribed to obtain a nucleic acid sequence encoding the protein marker (SEQ ID NO.8).

[0135] In the protein marker (referred to as 15kDa-WT) with the amino acid sequence as SEQ ID NO.7, the thiol group was selected as the target reactive group. The groups that can participate in the reaction in the amino acid sequence of this protein are the N-terminal amino group, the C-terminal hydroxyl group, and the side chain thiol group of cysteine. In order to avoid the occurrence of side reactions, the highly reactive lysine, aspartic acid, and glutamic acid in the protein were mutated into amino acids that do not participate in the reaction: Lysine K 6 , K 21 , K 55 , K 72 , K 85 All mutations to arginine R, i.e. K 6 R.K 21 R.K 55 R.K 72 R.K 85 R, glutamate E 99 、E 104 、E 112 All mutations to glutamine Q, ie E 99 Q.E 104 Q.E 112 Q, the purpose of this modification is to inhibit its reaction by controlling pH. In order to reduce the influence of steric effect in nucleophilic reaction and ensure the clarity and sharpness of protein modification, the positions of two adjacent target amino acids in the protein are adjusted: one of the two adjacent target amino acids is mutated, that is, C 35 GPCKMI 41Mutation to C 35 GPMCRI 41 The amino acid sequence of the modified protein marker (abbreviated as 10kDa-MUT) is shown in SEQ ID NO. 9. The sequence comparison results of the unmodified protein marker (abbreviated as 15kDa-WT) and the modified protein marker (abbreviated as 15kDa-MUT) are shown in Figure 7 .

[0136] The protein marker (abbreviated as 15kDa-MUT) obtained by transformation with the amino acid sequence shown in SEQ ID NO.7 was reverse transcribed to obtain a nucleic acid sequence encoding the protein marker (SEQ ID NO.10).

[0137] The target gene was synthesized according to the nucleic acid sequence of 15kDa-WT and 15kDa-MUT, and the prestained protein marker 15kDa-WT and prestained protein marker 15kDa-MUT were prepared in the same manner as the unmodified protein marker (referred to as 10kDa-WT). The difference was that since the thiol group was selected as the target reaction group, the reaction pH was adjusted to 8.5 (i.e., sodium carbonate-sodium bicarbonate buffer at pH 8.5).

[0138] Take the prestained protein Marker 15kDa-WT and prestained protein Marker 15kDa-MUT for SDS-PAGE to detect the effect of the 15kDa-WT band and the prestained protein Marker 15kDa-MUT conditions in electrophoresis. The results are as follows Figure 8-Figure 9 As shown in the figure, after 15kDa-WT was modified by dye coupling, the effect was poor, and the phenomenon on SDS-PAGE electrophoresis was that the protein bands were not sharp, the clarity was low, and even diffuse tailing. After 15kDa-MUT was modified by dye coupling, the bands were sharp and bright in color, meeting the target requirements. It can be seen that the modification of the primary structure reaction groups of the protein, the mutation of the highly reactive non-target amino acids, the reaction efficiency of the protein was guaranteed at the optimal pH and dye input, and the purpose of controlling the band shape of the modified protein was achieved, so that the target molecular weight protein can be obtained quickly and accurately.

[0139] 2. Take the protein marker with amino acid sequence as shown in SEQ ID NO.11 as an example

[0140] The protein marker (abbreviated as 25kDa-WT) with an amino acid sequence as shown in SEQ ID NO.11 was reverse transcribed to obtain a nucleic acid sequence encoding the protein marker (SEQ ID NO.12).

[0141] In the protein marker (referred to as 25kDa-WT) with an amino acid sequence such as SEQ ID NO.11, the amino group was selected as the target reactive group. The groups that can participate in the reaction in the amino acid sequence of this protein are the N-terminal amino group, the C-terminal hydroxyl group, and the lysine. In order to avoid the occurrence of side reactions, the highly reactive cysteine, aspartic acid, and glutamic acid in the protein were mutated into amino acids that do not participate in the reaction: cysteine ​​C 57 , C 60 , C 81 , C 173 , C 176 All mutations to arginine M, i.e. C 57 M.C 60 M.C 81 M.C 173 M.C 176 M; Glutamate E 22 、E 56 、E 862 、E 138 、E 171 All mutations to glutamine Q, ie E 22 Q.E 56 Q.E 862 Q.E 138 Q.E 171 Q; Aspartic acid D 17 , D 45 , D 93 , D 101 , D 133 All mutations to asparagine N, i.e. D 17 N, D 45 N, D 93 N, D 101 N, D 133 N, the purpose of this modification is to inhibit its reaction by controlling pH. In this protein, the hydrophobicity of the region where the target amino acid K is located is relatively weak, and no adjustment is made. The amino acid sequence of the modified protein marker (referred to as 25kDa-MUT) is shown in SEQ ID NO.13. The sequence alignment results of the unmodified protein marker (referred to as 25kDa-WT) and the modified protein marker (referred to as 25kDa-MUT) are shown in Fig.10 .

[0142] The protein marker (25 kDa-MUT for short) obtained by transformation with the amino acid sequence shown in SEQ ID NO.11 was reverse transcribed to obtain a nucleic acid sequence encoding the protein marker (SEQ ID NO.14).

[0143] The target gene was synthesized according to the nucleic acid sequence of 25kDa-WT and 25kDa-MUT, and the prestained protein marker 25kDa-WT and prestained protein marker 25kDa-MUT were prepared according to the same method as the unmodified protein marker (referred to as 10kDa-WT). The difference was that since the amino group was selected as the target reaction group, the reaction pH was adjusted to 10.0 (i.e., sodium carbonate-sodium bicarbonate buffer with pH 10.0).

[0144] Take the prestained protein Marker 25kDa-WT and prestained protein Marker 25kDa-MUT for SDS-PAGE and Western Bolt to detect the effects of the 25kDa-WT band and the prestained protein Marker 25kDa-MUT conditions in electrophoresis and immunoblotting experiments. The results are as follows: Figure 11-12 As shown in the figure, after 25kDa-WT was modified by dye coupling, the effect was poor, and the phenomenon on SDS-PAGE electrophoresis was that the protein bands were not sharp, the clarity was low, and even diffuse tailing. After 25kDa-MUT was modified by dye coupling, the bands were sharp and bright in color, meeting the target requirements. It can be seen that the modification of the primary structure reaction groups of the protein, the mutation of the highly reactive non-target amino acids, the reaction efficiency of the protein was guaranteed at the optimal pH and dye input, and the purpose of controlling the band shape of the modified protein was achieved, so that the target molecular weight protein can be obtained quickly and accurately.

[0145] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing a prestained protein marker, characterized in that: The method comprises: performing protein modification on a protein marker to obtain a modified protein marker; mixing a dye with the modified protein marker and performing a nucleophilic addition reaction or a nucleophilic substitution reaction to obtain a pre-stained protein marker; The protein modification of the protein marker comprises: using a group on an amino acid that can undergo a nucleophilic addition or nucleophilic substitution reaction with a dye as a reactive group, selecting any one of the reactive groups as a target group, and the remaining reactive groups as non-target groups; Amino acids with reactive groups are used as reactive amino acids, and amino acids without reactive groups are used as non-reactive amino acids. Among the reactive amino acids, amino acids with target groups are used as target amino acids, and amino acids with non-target groups are used as non-target amino acids. The non-target amino acids in the protein marker are blocked, or the non-target amino acids in the protein marker are mutated into non-reactive amino acids to obtain a modified protein marker.

2. A prestained protein marker, characterized in that: The prestained protein marker is prepared by the method according to claim 1.

3. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis method, characterized in that: The electrophoresis method is not for the purpose of disease diagnosis and treatment, and the electrophoresis method uses the prestained protein marker described in claim 2.

4. A protein immunoblotting experimental method, characterized in that: The experimental method is not for the purpose of disease diagnosis and treatment, and the experimental method uses the prestained protein marker described in claim 2.

5. A protein modification method for preparing a prestained protein marker, characterized in that: The protein modification method comprises: using groups on amino acids that can undergo nucleophilic addition or nucleophilic substitution reactions with dyes as reactive groups, selecting any one of the reactive groups as a target group, and the remaining reactive groups as non-target groups; Amino acids with reactive groups are used as reactive amino acids, and amino acids without reactive groups are used as non-reactive amino acids. Among the reactive amino acids, amino acids with target groups are used as target amino acids, and amino acids with non-target groups are used as non-target amino acids. The non-target amino acids in the protein marker are blocked, or the non-target amino acids in the protein marker are mutated into non-reactive amino acids to obtain a modified protein marker.

6. A modified protein for preparing a prestained protein marker, characterized in that: The modified protein is obtained by the protein modification method according to claim 5.

7. A nucleic acid molecule for preparing a prestained protein marker, characterized in that: The nucleic acid molecule encodes the engineered protein according to claim 6.

8. A recombinant plasmid for preparing a prestained protein marker, characterized in that: The recombinant plasmid expresses the modified protein according to claim 6.

9. A host cell for preparing a prestained protein marker, characterized in that: The host cell expresses the engineered protein according to claim 6.

10. Use of the method according to claim 1 or the prestained protein marker according to claim 2 or the protein modification method according to claim 5 or the modified protein according to claim 6 or the nucleic acid molecule according to claim 7 or the recombinant plasmid according to claim 8 or the host cell according to claim 9 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis or protein immunoblotting experiments, characterized in that: The application is not for the purpose of diagnosis and treatment of diseases.

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