CN114791458A - Gel for separating protein through electrophoresis, buffer solution used in cooperation with gel and kit of gel - Google Patents

Abstract

The invention belongs to the field of protein electrophoresis reagents, and discloses a gel for separating proteins by electrophoresis, a buffer solution used in combination and a kit thereof. Including gels containing gel buffers, gel system stabilizers, protein solubilizers, acrylamide and gels containing 2,2-bis-dimethylolaniline or trimethylolaminomethane, 3-morpholine propanesulfonic acid ( MOPS) or 2-N-morpholinoalkanesulfonic acid (MES), sodium dodecyl sulfate and EDTA gel electrophoresis buffers and gel electrophoresis kits containing the gels and gel electrophoresis buffers described above . The present invention can reduce the amount of the main buffer to 100mM-150mM, and the addition of glutamic acid in the present invention can prevent the accumulation of hydrogen ions at the bottom of the gel, thereby avoiding that the local pH of the gel is lower than 2 and the protein cannot migrate.

Description

A kind of gel for separating protein by electrophoresis, buffer solution used in combination and kit thereof

technical field

The invention belongs to the field of protein electrophoresis reagents, and discloses a gel for separating proteins by electrophoresis, a buffer solution used in combination and a kit thereof.

Background technique

Polyacrylamide gel electrophoresis is mainly used to separate proteins and nucleic acids, and is widely used in biology, medicine and other biochemical related fields. By changing the concentration of acrylamide before polymerization, polyacrylamide gels with different pore sizes can be prepared. In 1970, laemmli created a denaturing polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE), referred to as the laemmli system gel, which is widely used in the analysis of proteins.

The traditional laemmli system uses Tris as a buffer to control the pH of the gel, and the pH of the lower gel is 8.8. The laemmli system gel has good separation effect and band definition. However, because of the alkaline environment in the gel, the polyacrylamide after polymerization is easily hydrolyzed, which will disturb the migration of proteins during electrophoresis, and make protein bands. With blurry.

When the pH of the gel is adjusted to be slightly acidic, the stability of the polyacrylamide gel is greatly improved, and the gel can be prepared in advance, which greatly saves the operation time of protein electrophoresis experiments. Improves the stability of protein electrophoresis results.

Because its core is to control its pH value in a slightly acidic environment, the currently used buffers are mainly bis(2-hydroxymethyl)amino-tris(hydroxymethyl)methane and tris(hydroxymethyl)aminomethane. In electrophoresis, the types of trailing ions have a great influence on electrophoresis, and are mainly divided into traditional glycine and zwitterion buffers (Good's buffer). When using the above three, compared to the laemmli system, the electrophoretic migration speed is faster, which saves time, and the probability of protein modification during electrophoresis is lower, but the gel of this system is separated. The systems are quite different, and the prices of the raw materials are high.

During electrophoresis, the anode area will accumulate hydrogen ions with electrophoresis. The role of bis(2-hydroxymethyl)amino-tris(hydroxymethyl)methane and tris(hydroxymethyl)aminomethane in the two buffers is to ensure coagulation. The pH value in the gel is stable. If the buffer capacity is insufficient, the hydrogen ions cannot be absorbed, and the pH value in the area near the anode at the bottom of the gel is greatly reduced, resulting in the inability of the protein to migrate in the lower part of the gel. Therefore, the existing method cannot reduce the cost by reducing the concentration of the buffer.

When electrophoresis in the precast gel system, due to the low pH value compared with the traditional laemmli system, the actual load during electrophoresis is lower than that of the traditional method, and when separating proteins by electrophoresis, sometimes there are particles that cannot affect the migration of proteins during electrophoresis.

SUMMARY OF THE INVENTION

In view of the above situation, the present invention discloses a gel for separating proteins by electrophoresis, a buffer solution used together with them, and a kit thereof.

The technical scheme of the present invention is as follows:

A gel for separating proteins by electrophoresis, comprising the following components:

Gel buffer: 2,2-bis-dimethylolaniline or tris-hydroxymethylaminomethane;

Gel system stabilizer: one or more of glutamic acid, citric acid, malic acid, and succinic acid;

Protein solubilizer: dimethylformamide;

Acrylamide: N,N-Methylenebisacrylamide.

Further, the gel for separating proteins by electrophoresis is obtained by catalysts: ammonium persulfate and TEMED.

Further, the gel buffer: 2,2-bis(dimethylol)aniline or tris(hydroxymethylaminomethane) has a component concentration of 100-400mM, preferably 100mM-150mM.

Further, the gel system stabilizer is glutamic acid, citric acid, malic acid, and succinic acid in any proportion, and the component concentration is 1-50 mM.

Further, the acrylamide: N,N-Methylenebisacrylamide, the component concentration is 700mM-3000mM.

Further, the component concentration of the protein solubilizer: dimethylformamide is 1mM-100mM.

Further, the pH value of the gel is 6.4-6.6.

Gel running buffer for use with the above-mentioned separation of proteins by electrophoresis, including the following components:

1. 2,2-bis(dimethylol)aniline or tris(hydroxymethyl)aminomethane, the component concentration is 10-100mM;

2. 3-morpholinopropanesulfonic acid (MOPS) or 2-N-morpholinoalkanesulfonic acid (MES), the component concentration is 10-100mM;

3. Sodium dodecyl sulfate, the component concentration is 1-5mM;

4. EDTA, the component concentration is 0.1-3 mM.

Further, a gel electrophoresis kit comprises the above-mentioned gel and gel electrophoresis buffer mixed in any proportion.

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

Because the buffering agent needs to control the pH value of the gel, it needs a certain concentration or more. The present invention adds an acidic amino acid (glutamic acid) in the existing formula, and the glutamic acid improves the buffering capacity of the gel in the acidic region and prevents the occurrence of acidification. At the same time, the addition of glutamic acid will also improve the stability of the gel; the buffer of glutamic acid itself is about pH 3, and the addition of glutamic acid can bind hydrogen ions through its carboxyl and amino groups to prevent the increase of hydrogen ions in the above. Ions accumulate, and at the same time, the amide bond of polyacrylamide itself will be hydrolyzed into carboxyl groups, so adding glutamic acid will also play a reverse inhibitory effect. Because the pH value of the gel is neutral or affected by the trailing ions, the electrophoretic separation protein load is low, and the tolerance to samples with intolerant particles is poor, so in the present invention, dimethylformamide is added to improve the dissolution of the sample. properties, thereby increasing protein loading and preventing insoluble particles from clogging the gel. Dimethylformamide is an excellent solvent with a wide range of uses. It can be used as a good solvent for a variety of high molecular polymers. It is inferred that it can be used as a solubilizer for proteins in polyacrylamide gels, increasing the solubility of proteins during migration. .

Compared with the prior art, the present invention can reduce the amount of the main buffer to 100mM-150mM, and the addition of glutamic acid in the present invention can prevent the accumulation of hydrogen ions at the bottom of the gel, thereby avoiding that the local pH of the gel is lower than 2 and Makes proteins immobile (an intuitive indication of hydrogen ion accumulation is that bromophenol blue turns yellow, then becomes immobile, and deforms).

At the same time, the gel of the present invention can increase the solubility of the protein, avoid too many insoluble particles in the sample, and make the electrophoresis band more straight.

Description of drawings

Fig. 1 is the electrophoresis figure in embodiment 1;

Fig. 2 is the electropherogram in embodiment 2;

Fig. 3 is the electrophoresis figure in embodiment 3;

Fig. 4 is the separation linear relation diagram of formula 1 (prepared on the same day) in Example 4, wherein the abscissa is the logarithm value of protein migration molecular weight with the base 10, and the ordinate is the distance between the upper edge of the migration band and the lower edge of bromophenol blue distance value;

Fig. 5 is the separation linear relationship diagram of formula 1 in Example 4 (stored at 37°C for twelve days after preparation), wherein the abscissa is the logarithm of the protein migration molecular weight with the base 10, and the ordinate is the distance from the upper edge of the migration band The distance value of bromophenol blue at the lower edge;

Fig. 6 is the separation linear relation diagram of formula 2 (prepared on the same day) in Example 4, wherein the abscissa is the logarithm value of protein migration molecular weight with 10 as the base, and the ordinate is the distance between the upper edge of the migration band and the lower edge of bromophenol blue distance value;

Fig. 7 is the separation linear relationship diagram of formula 2 (preserved at 37°C for twelve days after preparation) in Example 4, wherein the abscissa is the logarithm of the protein migration molecular weight with the base 10, and the ordinate is the distance from the upper edge of the migration band The distance value of bromophenol blue at the lower edge;

Fig. 8 is the separation linear relationship diagram of formula three (prepared on the same day) in Example 4, wherein the abscissa is the logarithm value of protein migration molecular weight with 10 as the base, and the ordinate is the distance between the upper edge of the migration band and the lower edge of bromophenol blue distance value;

Fig. 9 is the separation linear relationship diagram of formula 3 in Example 4 (stored at 37°C for twelve days after preparation), wherein the abscissa is the logarithm of the protein migration molecular weight with the base 10, and the ordinate is the distance from the upper edge of the migration band The distance value for bromophenol blue at the lower edge.

Detailed ways

The test methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

In this example, the polyacrylamide gel added with glutamic acid and dimethylformamide in the present invention is compared with the polyacrylamide gel (formulas reported in other patents or published in literature) that are not within the scope of the present invention. Separation was performed on the second day of gel preparation.

Use a glass plate or a plastic plate to prepare an aqueous solution of polyacrylamide concentration of 12% (T=12%, C=2.6%, C=N, the mass of N-methylenebisacrylamide/mass of acrylamide+N,N-methylenebisacrylamide Amide mass, N,N methylidene bisacrylamide concentration is 1688 mM). The gel solution contains density agent glycerol with a concentration of 8%, and the pH is adjusted to 6.4-6.6 with hydrochloric acid. Ammonium persulfate and TEMED are used as catalysts to catalyze the polymerization and cross-linking of N,N-methylenebisacrylamide to form a three-dimensional network. Polyacrylamide gel. The control solution is used to prepare polyacrylamide gel with the same concentration (the catalytic part is a conventional method or a technique known in the art, which is not particularly limited in the present invention). The specific formula is shown in Table 1 below.

Table 1: Formulation of Example 1

Electrophoresis was performed using a Mini-Protean electrophoresis tank, and the test electrophoresis buffer was formulated as 50 mM tris, 50 mM 3-morpholinopropanesulfonic acid, 0.1% SDS and EDTA. The isolated samples were samples of different concentrations prepared from mouse liver tissue. As shown in Fig. 1, comparing the obtained electropherograms, the patent formula (left in Fig. 1) has a straighter band shape at high concentration, and the intolerant particles accumulated due to too high concentration will not appear in the band.

Example 2

In this example, the polyacrylamide gel added with glutamic acid and dimethylformamide in the present invention is compared with the polyacrylamide gel that is not within the scope of the present invention (formulations reported in other patents, or formulas published in literature). Tested after 12 days at 37°C after formulation.

A glass or plastic plate was used to prepare a 12% polyacrylamide concentration in water (T=12%, C=2.6%, N,N methylenebisacrylamide at 1688 mM). The gel solution contains density agent glycerol with a concentration of 8%, and the pH is adjusted to 6.4-6.6 with hydrochloric acid. Ammonium persulfate and TEMED are used as catalysts to catalyze the polymerization and cross-linking of N,N-methylenebisacrylamide to form a three-dimensional network. Polyacrylamide gel. The control solution is used to prepare polyacrylamide gel with the same concentration (the catalytic part is a conventional method or a technique known in the art, which is not particularly limited in the present invention). The specific formula is shown in Table 2 below:

Table 2: Formulation of Example 2

Electrophoresis was performed using a Mini-Protean electrophoresis tank, and the test electrophoresis buffer was formulated as 50 mM tris, 50 mM 3-morpholinopropanesulfonic acid, 0.1% SDS and EDTA. The isolated samples were samples of different concentrations prepared from mouse liver tissue. Comparing the obtained electropherograms according to Fig. 2, the patent formula (right in Fig. 2) does not turn yellow after electrophoresis to the bottom of the gel (shown as the color of bromofinland turns yellow) after being placed for a long time, while the control gel (Fig. 2) left) will turn yellow at the bottom, making the bottom result unobtainable.

Example 3

In this example, the alternative stabilizer malic acid added by the present invention, the polyacrylamide gel with glutamic acid and dimethylformamide added at the same time, was placed at 37 degrees Celsius for 12 days after preparation, and the same formula gel was prepared on the same day. Compare.

A glass or plastic plate was used to prepare a 12% polyacrylamide concentration in water (T=12%, C=2.6%, N,N methylenebisacrylamide at 1688 mM). The gel solution contains density agent glycerol with a concentration of 8%, and the pH is adjusted to 6.4-6.6 with hydrochloric acid. Ammonium persulfate and TEMED are used as catalysts to catalyze the polymerization and cross-linking of N,N-methylenebisacrylamide to form a three-dimensional network. Polyacrylamide gel. The control solution is used to prepare polyacrylamide gel with the same concentration (the catalytic part is a conventional method or a technique known in the art, which is not particularly limited in the present invention). The specific formula is shown in Table 3:

Table 3: Formulation of Example 3

Electrophoresis was performed using a Mini-Protean electrophoresis tank, and the test electrophoresis buffer was formulated as 50 mM tris, 50 mM 3-morpholinopropanesulfonic acid, 0.1% SDS and EDTA. The isolated samples were prepared from mouse liver tissue with different concentrations, pre-stained markers, non-pre-stained markers and E. coli lysates. The obtained electropherograms are compared as shown in Figure 3. The left gel in Figure 3 is the gel prepared on the same day, and the right is the result after being placed at 37 degrees Celsius for 12 days. Except for some bands that are slightly dispersed, there is no obvious change.

Example 4

This example is also a thermally accelerated test to investigate the effect of using different combinations and concentrations of buffers and stabilizers within the scope of the present invention on polyacrylamide gel stability, especially band mobility. According to the gel formulation in the table below, use a glass plate or a plastic plate to prepare an aqueous solution of polyacrylamide concentration of 10% (T=12%, C=2.6%), and the concentration of N,N-methylenebisacrylamide is 1407mM. The gel solution contains density agent glycerol with a concentration of 8%, and the pH is adjusted to 6.4-6.6 with hydrochloric acid. Ammonium persulfate and TEMED are used as catalysts to catalyze the polymerization and cross-linking of N,N-methylenebisacrylamide to form a three-dimensional network. Polyacrylamide gel. The control solution is used to prepare polyacrylamide gel with the same concentration (the catalytic part is a conventional method or a technique known in the art, which is not particularly limited in the present invention).

recipe name distinguishing components Recipe A 200Mm Bis-Tris; 30Mm Glutamate; 10mM Dimethylformamide Recipe B 300Mm Tris; 30Mm Glutamate; 10mM DMF Recipe C 300Mm Tris; 30Mm Glutamate; 50mM DMF

Electrophoresis was performed using a Mini-Protean electrophoresis tank, and the test electrophoresis buffer was formulated as 50 mM tris, 50 mM 3-morpholinopropanesulfonic acid, 0.1% SDS and EDTA. The separated samples are non-prestained protein molecular weight standards. The above formula was placed at 37 degrees Celsius for 12 days after preparation, and the migration of non-prestained protein molecular weight standards in gels with different formulations and storage times was compared with the gel prepared on the day of use. position, the protein molecular weight mobility formula is: lgMr=-b*mR+K. Because the gel separation range is linear, but a concentration only has a linear relationship within a certain protein molecular weight separation range, for example, a 10% concentration gel has a linear relationship within the protein molecular mass range of 40-70KDa. The results are shown in Figures 4-9. The migration positions of protein markers are basically the same for different composition ratios and storage times.

It will be apparent to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, but that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments are to be regarded in all respects as illustrative and not restrictive, and the scope of the invention is to be defined by the appended claims rather than the foregoing description, which are therefore intended to fall within the scope of the claims. All changes within the meaning and range of the equivalents of , are included in the present invention.

In addition, it should be understood that although this specification is described in terms of embodiments, not each embodiment only includes an independent technical solution, and this description in the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole , the technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.

Claims (10)

1. A gel for separating proteins by electrophoresis, the gel comprising:

gel buffer: 2, 2-bis-dimethylolaniline or tris (hydroxymethyl) aminomethane;

gel system stabilizer: one or more of glutamic acid, citric acid, malic acid and succinic acid;

protein solubilizer: dimethylformamide;

acrylamide: n, N-methylene bisacrylamide.

2. The gel for separating proteins by electrophoresis as claimed in claim 1, wherein the gel is prepared by contacting the protein with a catalyst: ammonium persulfate and TEMED.

3. The gel for separating proteins by electrophoresis as recited in claim 1, wherein said gel buffer: the component concentration of 2, 2-bis-dimethylolaniline or tris (hydroxymethyl) aminomethane was 100-400 mM.

4. The gel for separating proteins by electrophoresis as claimed in claim 1, wherein the gel system stabilizer is glutamic acid, citric acid, malic acid, succinic acid at any ratio, and the concentration of the components of the gel system stabilizer is 1-50 mM.

5. The gel for separating proteins by electrophoresis as claimed in claim 1, wherein the protein solubilizing agent: the concentration of the component dimethyl formamide is 1mM-100 mM.

6. The gel for separating proteins by electrophoresis as claimed in claim 1, wherein the acrylamide; the component concentration of the N, N-methylene bisacrylamide is 700mM-3000 mM.

7. The gel for separating proteins by electrophoresis as claimed in claim 1, wherein the pH of the gel is 6.4-6.6.

8. A gel electrophoresis buffer for use with a gel for separating proteins by electrophoresis according to any one of claims 1 to 7 comprising:

a first step of reacting 2, 2-bis-dimethylolaniline or tris (hydroxymethyl) aminomethane;

3-morpholinopropanesulfonic acid or 2-N-morpholinoalkanesulfonic acid;

③ sodium dodecyl sulfate;

④EDTA。

9. the gel electrophoresis buffer of claim 8 wherein the concentration of the 2, 2-bis-dimethylolaniline or tris is 10-100mM, the concentration of the 3-morpholinopropanesulfonic acid or 2-N-morpholinoalkanesulfonic acid is 10-100mM, the concentration of the sodium dodecylsulfate is 1-5mM, and the concentration of the EDTA is 0.1-3 mM.

10. A kit for gel electrophoresis comprising the gel for separating proteins by electrophoresis according to any one of claims 1 to 7 and the gel electrophoresis buffer according to claim 7 mixed at an arbitrary ratio.

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