A high-purity reactive dye and its preparation method and its application in preparing pre-stained protein marker

Abstract

The invention discloses a high-purity active dye and a preparation method thereof and an application thereof in preparing a pre-stained protein marker. The dye matrix is ​​first salted out with saturated sodium chloride, and then a para-ester diazonium salt is added to fully couple the reaction to obtain a high-purity active dye. The invention does not require repeated salting out and purification, reduces the problem of excessive salt content caused by repeated salting out and purification, and then uneven dyeing, improves the diazotization efficiency, improves the purity of the dye, concentrates the band of the pre-stained protein marker, and stabilizes the color. The vinyl sulfone type active dye and the amino group on the protein undergo affinity addition, and the protein electrophoresis band after dyeing is stable, the color is saturated, and the color is sharp and concentrated without dispersion.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a high-purity active dye and a preparation method thereof and application of the dye in preparing a pre-stained protein marker. Background Art

[0002] Reactive dyes are also called reactive dyes. They have one or more active groups in their molecular structure. Under certain conditions, they can form stable covalent bonds with cellulose, protein, and polyamide. Therefore, they have good wet fastness and are often used for textile dyeing. Reactive dyes have a wide range of sources. By regulating the combination of the matrix and the active groups, various chromatographic dyes can be constructed. They have become the main dye variety due to their bright colors, low cost, and convenient application.

[0003] Prestained protein marker is a mixture of prestained proteins of a specific molecular weight, which is used as a protein electrophoresis indicator in western blot experiments. Reactive dyes are the upstream raw materials of prestained protein markers, and their quality has a great impact on the quality of protein markers. In the traditional method of synthesizing reactive dyes, the dyes are usually purified by salting out in the last step, which inevitably introduces more salt into the final product. Reactive dyes with high salt content may cause protein precipitation during the process of dyeing high-concentration proteins, resulting in uneven protein dyeing. On the other hand, in order to improve color fastness, most reactive dyes on the market contain multiple reactive groups, and dyes containing multiple reactive groups are not suitable for dyeing proteins of specific molecular weights. Multiple reactive groups may construct covalent bonds between different protein molecules, resulting in multiple different molecular weight bands. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a reactive dye for pre-staining protein markers and a preparation method thereof.

[0005] The first object of the present invention is to provide a method for preparing a high-purity reactive dye.

[0006] The second object of the present invention is to provide a high-purity reactive dye prepared by the preparation method.

[0007] The third object of the present invention is to provide the use of the high-purity reactive dye in preparing pre-stained protein markers.

[0008] The fourth object of the present invention is to provide a prestained protein marker.

[0009] In order to achieve the above object, the present invention is implemented by the following scheme:

[0010] A method for preparing a high-purity reactive dye comprises firstly using saturated sodium chloride to salt out the dye matrix, then adding a para-ester diazonium salt, and fully coupling the reaction to obtain a high-purity reactive dye.

[0011] The chemical structural formula of the dye precursor is shown in Formula I, IV, VI or VIII:

[0012]

[0013] The para-ester diazonium salt is produced by diazotization reaction of the para-ester, concentrated hydrochloric acid and sodium nitrite;

[0014] The chemical structural formula of the para-ester is shown in Formula II:

[0015]

[0016] During the preparation process, the dye precursor is configured into a dye precursor aqueous solution with a pH of 5 to 6, and then salting out is performed.

[0017] Furthermore, the ratio of the dye precursor to water is 0.94-2 g:30 mL.

[0018] Furthermore, the pH of the parent aqueous solution is adjusted with sodium carbonate.

[0019] During the preparation process, the amino group of the dye precursor having the chemical structure shown in Formula IV or VI in the dye precursor aqueous solution needs to be blocked.

[0020] Furthermore, the amino group of the dye precursor having the chemical structure shown in Formula IV or VI is blocked with acetic anhydride.

[0021] During the amino blocking process, the temperature of the matrix aqueous solution is adjusted to 69-71°C, and then acetic anhydride is mixed and reacted to fully reach the blocking reaction endpoint to obtain a blocked reaction system. The blocked reaction system is then heated to boiling (under standard atmospheric pressure conditions) for 9.5-10.5 minutes to block the amino groups of the dye matrix and obtain an amino-blocked dye matrix.

[0022] In the preparation process, the para-ester, concentrated hydrochloric acid and sodium nitrite are mixed, and after sufficient diazotization reaction, the mixture is placed at -80 to -20°C for 5 to 15 minutes to precipitate the para-ester diazonium salt.

[0023] During the preparation process, the ratio of the para-ester, concentrated hydrochloric acid and sodium nitrite is 1.4-4.2 g: 5-10 mL: 1.5-5 mL, and it is ensured that all the amino groups on the para-ester are converted into diazonium salt.

[0024] Furthermore, the ratio of the para-ester, concentrated hydrochloric acid and sodium nitrite is 1.4 g: 5 mL: 1.5 mL, or 2.81 g: 10 mL: 2.5 mL, or 4.2 g: 10 mL: 5 mL.

[0025] During the preparation process, the concentration of sodium nitrite used can be 39-41% (mass concentration).

[0026] Furthermore, the concentration of the sodium nitrite used is 40% (mass concentration).

[0027] During the preparation process, the para-ester is first reacted with concentrated hydrochloric acid in an ice bath for 9 to 11 minutes, and then reacted with sodium nitrite for 29 to 31 minutes.

[0028] In a specific embodiment of the present invention, the para-ester is first reacted with concentrated hydrochloric acid in an ice bath for 10 minutes, and then reacted with sodium nitrite for 30 minutes.

[0029] During the preparation process, after being placed at -80 to -20°C for 5 to 15 minutes, the precipitated para-ester diazonium salt is filtered, rinsed with a saturated sodium chloride solution, and redissolved with water to obtain a para-ester diazonium salt solution. The ratio of water used to the para-ester used to prepare the para-ester diazonium salt is 20 mL: 1.4 to 4.2 g.

[0030] Furthermore, the ratio of the water used to the para-ester used to prepare the para-ester diazonium salt is 20 mL: 1.4 g, 20 mL: 2.81 g or 20 mL: 4.2 g.

[0031] During the preparation process, the pH is maintained at 5-6 and the temperature is less than 10° C. to fully carry out the coupling reaction and separate the solid and liquid to obtain the high-purity reactive dye.

[0032] The dye precursors with chemical structures such as Formula I, IV, VI or VIII react with the para-ester diazonium salt prepared from the para-ester with chemical structure such as Formula II to obtain reactive dyes with chemical structures such as Formula III, V, VII and IX respectively:

[0033]

[0034]

[0035] Furthermore, the coupling reaction time is 2 to 3 hours.

[0036] Furthermore, the molar ratio of the dye precursor with the chemical structure shown in Formula I to the para-ester with the chemical structure shown in Formula II is 1.5:1 to 1:1.5, and the mass ratio is 4.5g:2.81g to 2g:2.81g.

[0037] Furthermore, the molar ratio of the dye precursor with the chemical structure shown in Formula I to the para-ester with the chemical structure shown in Formula II is 0.64:1, and the mass ratio is 2g:2.81g.

[0038] Furthermore, the molar ratio of the dye precursor represented by the chemical formula IV to the para-ester represented by the chemical formula II is 1.5:1 to 1:1.5, and the mass ratio is 3.6 g:2.81 g to 1.6 g:2.81 g.

[0039] Furthermore, the molar ratio of the dye precursor with the chemical structure shown in Formula IV to the para-ester with the chemical structure shown in Formula II is 1.06:1, and the mass ratio is 3.9g:4.2g.

[0040] Furthermore, the molar ratio of the dye precursor with the chemical structure shown in Formula VI to the para-ester with the chemical structure shown in Formula II is 1.5:1 to 1:1.5, and the mass ratio is 2.82g:2.81g to 1.25g:2.81g.

[0041] Furthermore, the molar ratio of the dye precursor with the chemical structure shown in Formula VI to the para-ester with the chemical structure shown in Formula II is 1 to 1, and the mass ratio is 0.94 g:1.4 g.

[0042] Furthermore, the molar ratio of the dye precursor with the chemical structure shown in Formula VIII to the para-ester with the chemical structure shown in Formula II is 1.5:1 to 1:1.5, and the mass ratio is 3.48g:2.81g to 2.15g:2.81g.

[0043] Furthermore, the molar ratio of the dye precursor with the chemical structure shown in Formula VIII to the para-ester with the chemical structure shown in Formula II is 0.61:1, and the mass ratio is 2g:2.81g.

[0044] The present invention also claims protection for the high-purity reactive dye prepared by the preparation method.

[0045] The present invention also claims to protect the use of the high-purity reactive dye in preparing pre-stained protein markers.

[0046] The present invention also claims protection for a pre-stained protein marker obtained by staining the protein with the high-purity active dye.

[0047] In the embodiment of the present invention, the high-purity reactive dye is coupled with the protein to be stained and then purified by dialysis to obtain a prestained protein Marker.

[0048] Taking BSA protein as an example, the steps for preparing prestained protein marker are as follows:

[0049] S1: Prepare BSA protein into a 50 mg / mL test protein solution (solvent is 0.5 M carbonate buffer), and prepare the active dye into a 5 mg / mL active dye aqueous solution;

[0050] S2: Mix the reactive dye aqueous solution with the test protein solution in a volume ratio of 1 to 25:50, add 50% glycerol to a total volume of 110 μL, and carry out coupling reaction at 60°C for 50 min to obtain the stained protein.

[0051] S3: Dialysis step S2 After staining, the protein is dialyzed (10kd) to obtain the pre-stained protein marker.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] 1. Compared with the traditional dye synthesis method, the synthesis method of the present invention is simpler and easier to operate. The final product does not need to be purified by repeated salting out, which reduces the problem of excessive salt content caused by repeated salting out purification in the traditional method, thereby reducing the problem of uneven dyeing. The reaction time can be controlled within 3 to 4 hours to obtain a high-purity active dye.

[0054] 2. The present invention has strong applicability. Only a simple structural design or selection of raw materials available on the market can be used to obtain single reactive dyes for various types of chromatography, thereby improving the diazotization efficiency, improving the purity of the dye, concentrating the bands of the pre-stained protein marker, and stabilizing the color. The single active site group is conducive to the stabilization of the coupling reaction between the reactive dye and the protein of a specific molecular weight, thereby avoiding the appearance of multiple bands of different molecular weights.

[0055] 3. The single reactive dye synthesized by the present invention is particularly suitable for staining proteins of specific molecular weight. The vinyl sulfone type reactive dye undergoes affinity addition with the amino group on the protein. The protein electrophoresis band after staining is stable, the color is saturated, the color is sharp and concentrated, and does not diffuse. It is an ideal raw material for the production of pre-stained protein markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The SDS-PAGE electrophoresis results of the premixed protein marker prepared after different amounts of rose red dye staining BSA protein in Example 1. A is the direct electrophoresis picture, bands 1 to 6 correspond to the direct electrophoresis pictures of BSA stained with 1 μL, 5 μL, 10 μL, 15 μL, 20 μL and 25 μL rose red dye, respectively, and the band to the left of band 1 is a commercial protein marker; B is the picture after staining, bands 7 to 12 correspond to the pictures of BSA stained with rose red dye stained with bands 1 to 6, respectively, and the band to the left of band 7 is a commercial protein marker.

[0057] Figure 2The SDS-PAGE electrophoresis results of the premixed protein marker prepared after different amounts of orange dye-stained BSA protein in Example 2. A is the picture after direct electrophoresis, and bands 1 to 3 correspond to the pictures after direct electrophoresis of 15 μL, 20 μL and 25 μL orange dye-stained BSA mixed with homemade blue prestained protein, respectively. The band to the left of band 1 is a commercial protein marker, and bands 4 to 6 correspond to the pictures after direct electrophoresis of 15 μL, 20 μL and 25 μL orange dye-stained BSA, respectively; B is the picture after test dyeing, and bands 7 to 12 are the pictures after test dyeing corresponding to each band in A, and the band to the left of band 7 is a commercial protein marker.

[0058] Figure 3 The SDS-PAGE electrophoresis results of the premixed protein marker prepared after different amounts of yellow dye staining BSA protein. A is the picture after direct electrophoresis, and bands 1 to 6 of A correspond to the direct electrophoresis pictures of 1μL, 5μL, 10μL, 15μL, 20μL and 25μL yellow dye staining BSA; B is the picture after staining, and bands 7 to 12 correspond to the pictures of bands 1 to 6 of yellow dye staining BSA after staining.

[0059] Figure 4 The SDS-PAGE electrophoresis results of the premixed protein marker prepared after staining BSA protein with different amounts of fluorescent yellow dye. A is the direct electrophoresis image, and bands 1 to 6 correspond to the direct electrophoresis images of 1μL, 5μL, 10μL, 15μL, 20μL and 25μL of fluorescent yellow dye-stained BSA; B is the photo after dyeing, and bands 7 to 12 correspond to the photo after dyeing of bands 1 to 6 of fluorescent yellow dye-stained BSA.

[0060] Figure 5 The SDS-PAGE electrophoresis results of the premixed protein marker prepared by staining the BSA protein with the orange reactive dye prepared in Example 5 (bands 1 to 4, bands 8 to 11) and the orange reactive dye prepared in Example 2 (bands 5 to 6, bands 12 to 13). A is the picture after direct electrophoresis, the left side of band 1 is the commercial protein marker, and band 7 is the unstained BSA; B is the picture after staining, the left side of band 8 is the commercial protein marker, and band 14 is the unstained BSA.

[0061] Figure 6 The orange reactive dye ultraviolet absorption wavelength test of the orange reactive dye prepared in Example 2.

[0062] Figure 7 This is the liquid chromatogram of the orange reactive dye prepared in Example 2.

[0063] Figure 8This is the liquid chromatography purity data of the orange reactive dye prepared in Example 2.

[0064] Fig. 9 This is a liquid chromatogram of the orange reactive dye commercialized by Sigma.

[0065] Fig.10 The HPLC purity data for the commercial orange reactive dye from Sigma.

[0066] Fig.11 This is the liquid chromatogram of the yellow reactive dye prepared in Example 3.

[0067] Fig.12 This is the liquid chromatography purity data of the yellow reactive dye prepared in Example 3.

[0068] Fig.13 This is the liquid chromatogram of the rose red reactive dye prepared in Example 1.

[0069] Fig.14 This is the liquid chromatography purity data of the rose red reactive dye prepared in Example 1.

[0070] Fig.15 The SDS-PAGE electrophoresis results of the premixed protein marker after the BSA protein was stained with the commercial orange reactive dye of Sigma Company and the orange reactive dye prepared in Example 2 of the present invention. Wherein A is the commercial orange reactive dye of Sigma Company, and bands 1 to 6 correspond to 1 μL, 5 μL, 10 μL, 15 μL, 20 μL and 25 μL of Sigma dye stained BSA, and the left side of band 1 is the commercial protein marker; B is the orange reactive dye prepared in Example 2, and bands 7 to 12 correspond to 1 μL, 5 μL, 10 μL, 15 μL, 20 μL and 25 μL of Example 2 orange dye stained BSA. DETAILED DESCRIPTION

[0071] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0072] Example 1

[0073] 1. Experimental Methods

[0074] (1) Treatment of the precursor: Weigh 2 g of 8-anilino-1-naphthalenesulfonic acid magnesium salt (as shown in Formula I) in a 50 mL flask, add 30 mL of water, stir thoroughly to disperse, and obtain an aqueous solution of 8-anilino-1-naphthalenesulfonic acid magnesium salt. Carefully add sodium carbonate powder to the aqueous solution of 8-anilino-1-naphthalenesulfonic acid magnesium salt to adjust the pH until the pH of the aqueous solution of 8-anilino-1-naphthalenesulfonic acid magnesium salt reaches 5-6 and becomes clear, thereby obtaining a precursor solution.

[0075]

[0076] (2) Diazotization: Weigh 2.81g of para-ester (4-sulfoethyl sulfonylaniline, as shown in Formula II) and add it to a 150mL flask. Add 50g of crushed ice and stir thoroughly. Place in an ice bath, drop 10mL of concentrated hydrochloric acid and continue stirring for 10min. Add 2.5mL of 40% sodium nitrite solution and continue stirring for 30min. This is the diazotization reaction solution. Use Ehrlich to detect whether the diazotization reaction is complete. After the diazotization reaction is completed, remove the ice bath, and the diazotization reaction solution gradually returns to room temperature (20℃~25℃) and becomes clear. Transfer the diazotization reaction solution to -20℃ and place it for 15min or -80℃ and place it for 5min. A large amount of diazonium salt will precipitate. Filter it with suction, and carefully rinse it with saturated sodium chloride solution (25℃) several times to obtain a high-purity diazonium salt solid. Add 20mL of water to redissolve the diazonium salt solid to obtain a diazonium salt solution.

[0077]

[0078]

[0079] (3) Coupling reaction: Take a 500mL beaker, add 300mL saturated sodium chloride solution (25°C), add the parent solution prepared in step (1), and mix thoroughly. Slowly drop the diazonium salt solution in step (2), add sodium carbonate powder to adjust the pH to 5-6, and stir in an ice bath for 2-3h. After the reaction is completed, filter and obtain high-purity rose red reactive dye 8-(phenylamino)-7-((4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)diazenyl)naphthalene-1-sulfonic acid (as shown in Formula III).

[0080]

[0081] (4) Protein staining: Take 50 mg BSA protein and add it to 1 mL 0.5 M carbonate buffer (pH = 9.6) to obtain a test protein solution. Dilute the rose red reactive dye prepared in step (3) with water to form a 5 mg / mL rose red reactive dye aqueous solution. Take 1 μL, 5 μL, 10 μL, 15 μL, 20 μL and 25 μL of rose red reactive dye aqueous solution and vortex mix with 50 μL of the test protein solution, add 50% glycerol by volume to a total volume of 110 μL, and react at 60 ° C for 50 min to obtain the stained protein. The stained protein is dialyzed (10 kd) to obtain BSA protein stained with rose red reactive dye, which is used as a pre-stained protein Marker raw material. BSA protein is produced by Wuhan Saiweier Biotechnology Co., Ltd., item number GC305006-100g.

[0082] (5) SDS-PAGE electrophoresis: The BSA protein stained with different amounts of rose red reactive dye prepared in step (4) and a commercial protein marker (ThermoFisher brand) were subjected to SDS-PAGE electrophoresis and then photographed. The BSA protein was then stained with Coomassie Brilliant Blue staining solution and then photographed.

[0083] 2. Experimental Results

[0084] like Figure 1 As shown in A and B, the electrophoresis results of BSA protein stained with different amounts of rose red dye. After staining, the protein bands are concentrated and the color is stable. The effect of BSA staining with more than 10 μL of rose red dye is better.

[0085] Example 2

[0086] 1. Experimental Methods

[0087] (1) Treatment of the matrix: Weigh 3.9 g of gamma acid (as shown in Formula IV) into a 50 mL flask, add 30 mL of water, and stir thoroughly to disperse it to obtain a gamma acid aqueous solution. Carefully add sodium carbonate powder to the gamma acid aqueous solution to adjust the pH until the pH of the gamma acid aqueous solution reaches 5 to 6 and the solution becomes clear, thereby obtaining a pH-adjusted gamma acid aqueous solution.

[0088] Since γ-acid contains an amino group, it is necessary to block the amino group with acetic anhydride, otherwise the amino group on the parent body may also participate in the reaction during the diazotization reaction, resulting in an increase in the final by-products.

[0089] Heat the pH-adjusted gamma acid aqueous solution to 70°C to obtain a heated gamma acid aqueous solution. Take 2.3 mL of acetic anhydride and mix it in 5 mL of water to obtain an acetic anhydride aqueous solution. Slowly drop the acetic anhydride aqueous solution into the heated gamma acid aqueous solution, and use Ehrlich reagent to detect the end point of the blocking reaction. If the blocking reaction end point is not reached after the addition of the acetic anhydride aqueous solution is completed, continue to add the acetic anhydride aqueous solution until the blocking reaction is complete to obtain a blocked reaction system. After the blocking reaction is completed, heat the blocked reaction system to boiling (100-101 KPa) and keep it for 10 minutes, stop heating, and cool to room temperature (20°C-25°C).

[0090]

[0091] (2) Diazotization: The weight of the para-ester (4-ethylsulfatesulfonylaniline, as shown in Formula II) is 4.2 g, the volume of the 40% sodium nitrite solution is 5 mL, and the other reaction conditions are the same as those of the diazotization in step (2) of Example 1.

[0092]

[0093] (3) Coupling reaction: The coupling reaction is the same as that in step (3) of Example 1 to obtain a high-purity orange reactive dye:

[0094] (E)-6-acetamido-4-hydroxy-3-((4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)diazenyl)naphthalene-2-sulfonic acid (shown in Formula V).

[0095]

[0096] (4) Protein staining: The same as the protein staining in step (4) of Example 1, wherein the amount of orange reactive dye aqueous solution used was 15, 20 and 25 μL, to obtain BSA protein stained with orange reactive dye.

[0097] (5) SDS-PAGE electrophoresis:

[0098] The BSA protein stained with different amounts of orange reactive dye prepared in step (4) was subjected to SDS-PAGE electrophoresis and then photographed, and then stained with Coomassie Brilliant Blue staining solution and then photographed. A commercial marker was used as a control.

[0099] Different amounts of BSA protein stained with orange reactive dye prepared in step (4) were mixed with protein pre-stained with homemade blue dye, and photographed after SDS-PAGE electrophoresis, and then stained with Coomassie brilliant blue staining solution and photographed.

[0100] 2. Experimental Results

[0101] like Figure 2 The figure shows the electrophoresis effect of BSA protein stained with different amounts of orange reactive dye. After staining, the protein bands are concentrated and the color is stable. After mixing, the orange reactive dye staining of BSA protein is still stable.

[0102] Example 3

[0103] 1. Experimental Methods

[0104] (1) Treatment of the precursor: Weigh 0.94 g of 2,4-diaminobenzenesulfonic acid (as shown in Formula VI) into a 50 mL flask, add 30 mL of water, and stir thoroughly to disperse the mixture to obtain an aqueous solution of 2,4-diaminobenzenesulfonic acid.

[0105] Since 2,4-diaminobenzenesulfonic acid contains an amino group, it is necessary to block the amino group with acetic anhydride, otherwise the amino group on the parent body may also participate in the reaction during the diazotization reaction, resulting in an increase in the final by-products.

[0106] The 2,4-diaminobenzenesulfonic acid aqueous solution was heated to 70°C to obtain the heated 2,4-diaminobenzenesulfonic acid aqueous solution. 0.957 mL of acetic anhydride was slowly dripped into the heated 2,4-diaminobenzenesulfonic acid aqueous solution, and the end point of the blocking reaction was detected with Ehrlich reagent. If the blocking reaction end point is not reached after the addition of acetic anhydride is completed, acetic anhydride is continued to be added until the reaction is complete to obtain a blocking reaction system. After the blocking reaction is completed, the blocking reaction system is heated to boiling and maintained for 10 minutes, and the heating is stopped and cooled to room temperature.

[0107]

[0108] (2) Diazotization: The weight of the para-ester (4-sulfate ethyl sulfonylaniline, as shown in Formula II) is 1.4 g, the volume of the concentrated salt is 5 mL, the volume of the 40% sodium nitrite solution is 1.5 mL, and the other reaction conditions are the same as those of the diazotization in step (2) of Example 1.

[0109]

[0110] (3) Coupling reaction: The coupling reaction is the same as that in step (3) of Example 1 to obtain a high-purity yellow reactive dye:

[0111] 4-acetamido-2-amino-5-((4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)diazenyl)benzen esulfonic acid (as shown in Formula VII).

[0112]

[0113] (4) Protein staining: the same as the protein staining in step (4) of Example 1.

[0114] (5) SDS-PAGE electrophoresis: the same steps as in Example 1.

[0115] 2. Experimental Results

[0116] like Figure 3 The following is the electrophoresis effect of different amounts of yellow reactive dye staining BSA protein. After staining, the protein bands are concentrated and the color is stable. The effect of staining BSA with more than 10 μL of yellow reactive dye is better.

[0117] Example 4

[0118] 1. Experimental Methods

[0119] (1) Treatment of the precursor: Weigh 2 g of 1-(2,5-dichloro-4-sulfonate phenyl)-3-methyl-5-pyrazolone (as shown in Formula VIII) in a 50 mL flask, add 30 mL of water, stir thoroughly to disperse, and obtain an aqueous solution of 1-(2,5-dichloro-4-sulfonate phenyl)-3-methyl-5-pyrazolone. Carefully add sodium carbonate powder to the aqueous solution of 1-(2,5-dichloro-4-sulfonate phenyl)-3-methyl-5-pyrazolone to adjust the pH to 5-6, and the solution becomes clear to obtain a precursor solution.

[0120]

[0121] (2) Diazotization: The same as the diazotization in step (2) of Example 1, wherein the para-ester is (4-ethyl sulfate sulfonylaniline, as shown in Formula II).

[0122]

[0123] (3) Coupling reaction: The coupling reaction is the same as step (3) of Example 1 to obtain a high-purity fluorescent yellow active dye:

[0124] (E)-2,5-dichloro-4-(5-((2-methoxy-4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)diazen yl)-3,4-dimethyl-1H-pyrazol-1-yl)benzenesulfonic acid (as shown in Formula IX).

[0125]

[0126] (4) Protein staining: the same as step (4) protein staining in Example 1.

[0127] (5) SDS-PAGE electrophoresis: the same steps as in Example 1.

[0128] 2. Experimental Results

[0129] like Figure 4 The figure shows the electrophoresis effect of BSA protein stained with different amounts of fluorescent yellow active dye. After staining, the protein bands are concentrated and the color is stable. The effect of BSA staining with more than 10 μL of fluorescent yellow active dye is better.

[0130] Example 5

[0131] (1) Processing the matrix: the same as step (1) of Example 2.

[0132] (2) Diazotization: The same as the diazotization in step (2) of Example 2.

[0133] (3) Coupling reaction: slowly dropwise add the diazonium salt solution of step (2) to the parent solution of step (1), control the pH value between 5 and 6, and react in an ice bath for 2 to 3 hours. After the ice bath reaction, add 10 times the volume of saturated sodium chloride solution (25° C.) to the reaction system for recrystallization and purification, and filter with suction to obtain an orange reactive dye, the structural formula of which is the same as that of the orange reactive dye of Example 2.

[0134] (4) Protein staining: The protein staining was the same as that in step (4) of Example 1, wherein the amount of orange reactive dye aqueous solution used was 25 μL, to obtain BSA protein stained with orange reactive dye.

[0135] (5) SDS-PAGE electrophoresis: the same steps as in Example 1.

[0136] 2. Experimental Results

[0137] like Figure 5 As shown, the SDS-PAGE electrophoresis effect of the orange reactive dye (25 μL) prepared in Example 5 (bands 1 to 4 on the left side of the Marker) and the orange reactive dye (25 μL) prepared by the method of Example 2 (bands 5 to 6 on the left side of the Marker) for staining BSA protein, the protein bands of the orange reactive dye of Example 5 are not as concentrated as the protein bands of the orange reactive dye of Example 2.

[0138] Example 6

[0139] Experimental group 1: 4.5 g of the dye precursor with the chemical structure shown in Formula I in Example 1 was used, and other preparation conditions were the same as in Example 1 to obtain a rose red reactive dye.

[0140] Experimental Group 2: In Example 2, the dye precursor having the chemical structure shown in Formula IV is 3.6 g, the para-ester is 2.81 g, and the other preparation conditions are the same as in Example 2 to obtain an orange reactive dye.

[0141] Experimental Group 3: The dye precursor with the chemical structure shown in Formula IV in Example 2 is 1.6 g, the para-ester is 2.81 g, and the other preparation conditions are the same as in Example 2 to obtain an orange reactive dye.

[0142] Experimental Group 4: The dye precursor with the chemical structure shown in Formula VI in Example 3 is 2.82 g, the para-ester is 2.81 g, and the other preparation conditions are the same as in Example 3 to obtain a yellow reactive dye.

[0143] Experimental Group 5: The dye precursor with the chemical structure shown in Formula VI in Example 3 is 1.25 g, the para-ester is 2.81 g, and the other preparation conditions are the same as in Example 3 to obtain a yellow reactive dye.

[0144] Experimental Group 6: The dye precursor with the chemical structure shown in Formula VIII in Example 4 is 3.48 g, the para-ester is 2.81 g, and the other preparation conditions are the same as in Example 4 to obtain a fluorescent yellow active dye.

[0145] Experimental Group 7: The dye precursor with the chemical structure shown in Formula VIII in Example 4 is 2.15 g, the para-ester is 2.81 g, and the other preparation conditions are the same as in Example 4 to obtain a fluorescent yellow active dye.

[0146] The above experimental groups can obtain reactive dyes with good color effects.

[0147] Example 7

[0148] 1. Experimental Methods

[0149] Ultraviolet absorption detection: using an enzyme marker to test the absorption kinetics, the test sample's absorption value is between 270nm and 800nm.

[0150] Liquid chromatography detection: chromatographic column Welch Ultimate XB C18 250×4.6mm, 5μm; DAD detector, detection wavelengths at 384nm and 492nm; flow rate 1mL / min; column temperature 30℃.

[0151] Preparation of mobile phase: Mobile phase A is 20 nM sodium dihydrogen phosphate aqueous solution, mobile phase B is methanol, and the gradient shown in Table 1 is used for elution.

[0152] Table 1 Mobile phase gradient

[0153]

[0154]

[0155] 2. Experimental Results

[0156] like Figure 6As shown, the ultraviolet absorption wavelength test of the orange reactive dye prepared in Example 2 of the present invention shows that the maximum absorption peak is near 492 nm.

[0157] The orange reactive dye prepared in Example 2 of the present invention has a higher purity than the commercial orange reactive dye of Sigma Company. The liquid chromatography data and the chromatographic data show that the purity data are 97.8% ( Figure 7 and Figure 8 ) and 84.4% ( Fig. 9 and Fig.10 ).

[0158] The yellow reactive dye prepared in Example 3 of the present invention has a purity of up to 98% ( Fig.11 and Fig.12 ), the purity of the rose red reactive dye prepared in Example 1 of the present invention is as high as 99% ( Fig.13 and Fig.14 ).

[0159] Example 8

[0160] 1. Experimental Methods

[0161] Protein staining and SDS-PAGE electrophoresis were performed according to the method of Example 1: the amounts of the orange reactive dye prepared in Example 2 were 1 μL, 5 μL, 10 μL, 15 μL, 20 μL and 25 μL, respectively, and the amounts of the commercial orange reactive dye from Sigma were also the same.

[0162] 2. Experimental Results

[0163] like Fig.15 As shown, the protein bands of BSA stained with orange reactive dye prepared in Example 2 of the present invention are sharper and more concentrated, and not diffuse. However, the protein bands of BSA stained with orange reactive dye commercially produced by Sigma are not concentrated enough and have dragging marks.

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a high-purity reactive dye, characterized in that: First, use saturated sodium chloride to salt out the dye matrix, then add the para-ester diazonium salt to fully couple the reaction to obtain a high-purity reactive dye. The chemical structural formula of the dye precursor is shown in Formula I, IV or VI: The para-ester diazonium salt is produced by diazotization reaction of the para-ester, concentrated hydrochloric acid and sodium nitrite; during the preparation process, after being placed at -80 to -20°C for 5 to 15 minutes, the precipitated para-ester diazonium salt is filtered, rinsed with a saturated sodium chloride solution, and redissolved with water to obtain a para-ester diazonium salt solution, wherein the ratio of the water used to the para-ester used to prepare the para-ester diazonium salt is 20 mL: 1.4 to 4.2 g; The chemical structural formula of the para-ester is shown in Formula II: The dye matrix is ​​configured into a dye matrix aqueous solution with a pH of 5 to 6, and then salting out is performed.

2. The preparation method according to claim 1, characterized in that: The amino group of the dye precursor represented by the chemical formula IV or VI is blocked.

3. The preparation method according to claim 1, characterized in that: The ratio of the para-ester, concentrated hydrochloric acid and sodium nitrite is 1.4-4.2 g: 5-10 mL: 1.5-5 mL.

4. The preparation method according to claim 1, characterized in that: The molar ratio of the dye precursor with the chemical structural formula shown in Formula I, IV or VI to the para-ester with the chemical structural formula shown in Formula II is 1.5:1 to 1:1.

5.

5. The preparation method according to claim 1, characterized in that: The high-purity reactive dye is obtained by maintaining the pH value at 5-6 and the temperature at less than 10°C for a sufficient coupling reaction and solid-liquid separation.

6. Use of the high-purity reactive dye prepared by the preparation method according to any one of claims 1 to 5 in the preparation of pre-stained protein markers.

7. A prestained protein marker, characterized in that: The protein is obtained by using a high-purity active dye-dyed protein prepared by the preparation method described in any one of claims 1 to 5.

Images