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

Gel for separating protein through electrophoresis, buffer solution used in cooperation with gel and kit of gel Download PDF

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CN114791458A
CN114791458A CN202210440812.2A CN202210440812A CN114791458A CN 114791458 A CN114791458 A CN 114791458A CN 202210440812 A CN202210440812 A CN 202210440812A CN 114791458 A CN114791458 A CN 114791458A
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李广亮
李民强
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Dalian Bgbioscience Co ltd
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    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N27/447Systems using electrophoresis
    • G01N27/44756Apparatus specially adapted therefor
    • G01N27/44773Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
    • G01N27/44778Multi-stage electrophoresis, e.g. two-dimensional electrophoresis on a common gel carrier, i.e. 2D gel electrophoresis

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Abstract

本发明属于蛋白电泳试剂领域,公开了一种通过电泳分离蛋白的凝胶、配合使用的缓冲液及其试剂盒。包括含有凝胶缓冲剂、凝胶体系稳定剂、蛋白增溶剂、丙烯酰胺的凝胶和含有2,2‑双二羟甲基苯胺或三羟甲基氨基甲烷、3‑吗啉丙磺酸(MOPS)或2‑N‑吗啉代烷磺酸(MES)、十二烷基硫酸钠和EDTA的凝胶电泳缓冲液以及包含上述中的凝胶和凝胶电泳缓冲液的凝胶电泳试剂盒。本发明可将主要缓冲剂用量减少到100mM‑150mM,本发明中加入谷氨酸,可以防止凝胶的底部氢离子的蓄积,从而避免凝胶局部pH低于2而使蛋白质无法迁移。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

一种通过电泳分离蛋白的凝胶、配合使用的缓冲液及其试 剂盒A kind of gel for separating protein by electrophoresis, buffer solution used in combination and kit thereof

技术领域technical field

本发明属于蛋白电泳试剂领域,公开了一种通过电泳分离蛋白的凝胶、配合使用的缓冲液及其试剂盒。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.

背景技术Background technique

聚丙烯酰胺凝胶电泳主要用于分离蛋白质和核酸,在生物学、医学等生物化学相关领域都有广泛使用,通过改变聚合前丙烯酰胺的浓度,可以制备出不同孔径的聚丙烯酰胺凝胶。在1970年,laemmli创建了含有十二烷基硫酸钠的变性聚丙烯酰胺凝胶(SDS-PAGE),简称laemmli系统凝胶,这项技术被广泛应用于蛋白质的分析。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.

传统的laemmli系统由三羟甲基氨基甲烷作为缓冲剂控制凝胶pH值,下层胶的pH值为8.8。laemmli系统凝胶具有良好的分离效果和条带清晰度,然而因其凝胶内的碱性环境使聚合后的聚丙烯酰胺易被水解,会扰乱电泳过程中蛋白质的迁移,并会使蛋白条带模糊不清。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.

在将凝胶pH调整至偏酸性时,聚丙烯酰胺凝胶的稳定性得到极大提高,可以将凝胶提前制备好,极大的节省了蛋白电泳实验的操作时间,并因批量制备,也提高了蛋白电泳结果的稳定性。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.

因其核心是将其pH值控制在偏酸性环境,目前使用的缓冲剂主要是双(2-羟甲基)氨基-三(羟甲基)甲烷和三羟甲基氨基甲烷两类。在电泳中其尾随离子的种类对电泳影响很大,主要分为传统的甘氨酸和两性离子缓冲剂(Good’s buffer),Good’s buffer中作为电泳时尾随离子的主要是MOPS、MES和HEPES三种,在使用以上三种时,与laemmli系统相比,电泳迁移速度更快可节省时间,而且电泳过程中蛋白质被修饰的概率更低,但是这种系统的凝胶在分离时蛋白质的迁移位置与传统laemmli系统有较大不同,且原料的价格很高。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.

在电泳时其阳极区域会随电泳进行而蓄积氢离子,两种缓冲剂中双(2-羟甲基)氨基-三(羟甲基)甲烷和三羟甲基氨基甲烷的作用就是为保证凝胶内pH值的稳定,如果缓冲能力不够,会使氢离子无法得到吸收,使凝胶底部靠近阳极区域pH值大幅度降低,导致蛋白质在凝胶下部无法迁移。所以现有方法不能通过降低缓冲剂的浓度,来降低成本。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.

在预制胶体系内电泳时,因与传统laemmli系统相比pH值低,所以电泳时实际载量低于传统方法,且在电泳分离蛋白质时,有时会有不容颗粒影响电泳时蛋白质的迁移。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.

发明内容SUMMARY OF THE INVENTION

针对上述情况,本发明公开了一种通过电泳分离蛋白的凝胶、配合使用的缓冲液及其试剂盒。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.

本发明的技术方案如下:The technical scheme of the present invention is as follows:

一种通过电泳分离蛋白的凝胶,包括以下组份:A gel for separating proteins by electrophoresis, comprising the following components:

凝胶缓冲剂:2,2-双二羟甲基苯胺或三羟甲基氨基甲烷;Gel buffer: 2,2-bis-dimethylolaniline or tris-hydroxymethylaminomethane;

凝胶体系稳定剂:谷氨酸、柠檬酸、苹果酸、丁二酸中的一种或者多种;Gel system stabilizer: one or more of glutamic acid, citric acid, malic acid, and succinic acid;

蛋白增溶剂:二甲基甲酰胺;Protein solubilizer: dimethylformamide;

丙烯酰胺:N,N-甲叉双丙烯酰胺。Acrylamide: N,N-Methylenebisacrylamide.

进一步的,所述通过电泳分离蛋白的凝胶是通过催化剂:过硫酸铵和TEMED催化得到的。Further, the gel for separating proteins by electrophoresis is obtained by catalysts: ammonium persulfate and TEMED.

进一步的,所述凝胶缓冲剂:2,2-双二羟甲基苯胺或三羟甲基氨基甲烷的组分浓度为100-400mM,优选为100mM-150mM。Further, the gel buffer: 2,2-bis(dimethylol)aniline or tris(hydroxymethylaminomethane) has a component concentration of 100-400mM, preferably 100mM-150mM.

进一步的,所述凝胶体系稳定剂为任意配比的谷氨酸、柠檬酸、苹果酸、丁二酸,组分浓度为1-50mM。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.

进一步的,所述丙烯酰胺:N,N-甲叉双丙烯酰胺,组分浓度为700mM-3000mM。Further, the acrylamide: N,N-Methylenebisacrylamide, the component concentration is 700mM-3000mM.

进一步的,所述蛋白增溶剂:二甲基甲酰胺的组分浓度为1mM-100mM。Further, the component concentration of the protein solubilizer: dimethylformamide is 1mM-100mM.

进一步的,所述凝胶的pH值为6.4-6.6。Further, the pH value of the gel is 6.4-6.6.

与上述通过电泳分离蛋白凝胶配合使用的凝胶电泳缓冲液,包括以下组分:Gel running buffer for use with the above-mentioned separation of proteins by electrophoresis, including the following components:

1. 2,2-双二羟甲基苯胺或三羟甲基氨基甲烷,组分浓度为10-100mM;1. 2,2-bis(dimethylol)aniline or tris(hydroxymethyl)aminomethane, the component concentration is 10-100mM;

2. 3-吗啉丙磺酸(MOPS)或2-N-吗啉代烷磺酸(MES),组分浓度为10-100mM;2. 3-morpholinopropanesulfonic acid (MOPS) or 2-N-morpholinoalkanesulfonic acid (MES), the component concentration is 10-100mM;

3.十二烷基硫酸钠,组分浓度为1-5mM;3. Sodium dodecyl sulfate, the component concentration is 1-5mM;

4.EDTA,组分浓度为0.1-3mM。4. EDTA, the component concentration is 0.1-3 mM.

进一步的,一种凝胶电泳试剂盒,包含上述配比的凝胶和凝胶电泳缓冲液任意比例混合。Further, a gel electrophoresis kit comprises the above-mentioned gel and gel electrophoresis buffer mixed in any proportion.

与现有技术相比,本发明具有如下的有益效果:Compared with the prior art, the present invention has the following beneficial effects:

因缓冲剂需要控制凝胶pH值,所以需要一定浓度以上,本发明在现有配方内加入酸性氨基酸(谷氨酸),谷氨酸提高凝胶在酸性区域内的缓冲能力,防止酸化的发生;同时因谷氨酸的加入也会提高凝胶的稳定性;谷氨酸本身的缓冲区间在pH值3左右,加入谷氨酸可以通过其羧基和氨基结合氢离子,防止上文中提高的氢离子蓄积,同时聚丙烯酰胺本身的酰胺键会水解为羧基,所以加入谷氨酸也会起到逆向抑制作用。因凝胶的pH值是中性或受尾随离子的影响,导致电泳分离蛋白载量低,且对有不容颗粒的样品耐受性差,所以在本发明中加入二甲基甲酰胺,提高样品溶解性,从而提高蛋白载量,并能防止不溶颗粒堵塞凝胶。二甲基甲酰胺是用途很广的优良溶剂,可以作为多种高分子聚合物的良好溶剂,依次类推推测可用作聚丙烯酰胺凝胶中蛋白质的增溶剂,增加在迁移过程中蛋白质的溶解度。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. .

与现有技术相比,本发明可将主要缓冲剂用量减少到100mM-150mM,本发明中加入谷氨酸,可以防止凝胶的底部氢离子的蓄积,从而避免凝胶局部pH低于2而使蛋白质无法迁移(氢离子蓄积的直观表现是溴酚蓝会变黄,然后无法移动,并变形)。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).

同时,本发明所述的凝胶可以增加蛋白质的溶解度,避免样品中有过多的不溶颗粒,使得电泳条带更加平直。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.

附图说明Description of drawings

图1为实施例1中电泳图;Fig. 1 is the electrophoresis figure in embodiment 1;

图2为实施例2中的电泳图;Fig. 2 is the electropherogram in embodiment 2;

图3为实施例3中的电泳图;Fig. 3 is the electrophoresis figure in embodiment 3;

图4为实施例4中配方一(当天配制)分离线性关系图,其中横坐标为蛋白质迁移分子量以10为底的对数值,纵坐标是迁移条带的上边缘距离下边缘溴酚蓝的距离值;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;

图5为实施例4中配方一(配制后37℃保存十二天)分离线性关系图,其中横坐标为蛋白质迁移分子量以10为底的对数值,纵坐标是迁移条带的上边缘距离下边缘溴酚蓝的距离值;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;

图6为实施例4中配方二(当天配制)分离线性关系图,其中横坐标为蛋白质迁移分子量以10为底的对数值,纵坐标是迁移条带的上边缘距离下边缘溴酚蓝的距离值;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;

图7为实施例4中配方二(配制后37℃保存十二天)分离线性关系图,其中横坐标为蛋白质迁移分子量以10为底的对数值,纵坐标是迁移条带的上边缘距离下边缘溴酚蓝的距离值;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;

图8为实施例4中配方三(当天配制)分离线性关系图,其中横坐标为蛋白质迁移分子量以10为底的对数值,纵坐标是迁移条带的上边缘距离下边缘溴酚蓝的距离值;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;

图9为实施例4中配方三(配制后37℃保存十二天)分离线性关系图,其中横坐标为蛋白质迁移分子量以10为底的对数值,纵坐标是迁移条带的上边缘距离下边缘溴酚蓝的距离值。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.

具体实施方式Detailed ways

下述实施例中所使用的试验方法如无特殊说明,均为常规方法。The test methods used in the following examples are conventional methods unless otherwise specified.

下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径获得。Materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

实施例1Example 1

本实施例对本发明内添加了谷氨酸和二甲基甲酰胺的聚丙烯酰胺凝胶与不在本发明范围内的聚丙烯酰胺凝胶(其他专利报道配方、或文献公布公布配方)进行比较。在制备凝胶的第二天进行分离。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.

使用玻璃板或塑料板配制聚丙烯酰胺浓度12%的水溶液(T=12%,C=2.6%,C=N,N甲叉双丙烯酰胺的质量/丙烯酰胺质量+N,N甲叉双丙烯酰胺质量,N,N甲叉双丙烯酰胺浓度为1688mM)。制胶溶液中含有密度剂甘油浓度8%,并使用盐酸将pH调节至6.4-6.6之间,使用过硫酸铵和TEMED作为催化剂催化N,N甲叉双丙烯酰胺聚合交联形成三维网状的聚丙烯酰胺凝胶。对照溶液配制相同浓度的聚丙烯酰胺凝胶(催化部分为常规方法或本领域公知技术,本发明对此不进行特殊限定)。具体配方如下表1。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.

表1:实施例1的配方Table 1: Formulation of Example 1

Figure BDA0003614984960000051
Figure BDA0003614984960000051

使用Mini-Protean型电泳槽电泳,测试电泳缓冲液使用配方为50mM三羟甲基氨基甲烷、50mM 3-吗啉丙磺酸、0.1%SDS和EDTA。所分离样品为小鼠肝脏组织制备的不同浓度样品。如图1所示,对所得电泳图进行比较,专利配方(图1左)在高浓度时条带形状更加平直,因浓度过高蓄积的不容颗粒,不会在条带中显现。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.

实施例2Example 2

本实施例对本发明内添加了谷氨酸和二甲基甲酰胺的聚丙烯酰胺凝胶与不在本发明范围内的聚丙烯酰胺凝胶(其他专利报道配方、或文献公布公布配方)进行比较,在配制后37摄氏度放置12天后测试。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.

使用玻璃板或塑料板配制聚丙烯酰胺浓度12%的水溶液(T=12%,C=2.6%,N,N甲叉双丙烯酰胺浓度为1688mM)。制胶溶液中含有密度剂甘油浓度8%,并使用盐酸将pH调节至6.4-6.6之间,使用过硫酸铵和TEMED作为催化剂催化N,N甲叉双丙烯酰胺聚合交联形成三维网状的聚丙烯酰胺凝胶。对照溶液配制相同浓度的聚丙烯酰胺凝胶(催化部分为常规方法或本领域公知技术,本发明对此不进行特殊限定)。具体配方如下表2所示: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:

表2:实施例2的配方Table 2: Formulation of Example 2

Figure BDA0003614984960000061
Figure BDA0003614984960000061

使用Mini-Protean型电泳槽电泳,测试电泳缓冲液使用配方为50mM三羟甲基氨基甲烷、50mM 3-吗啉丙磺酸、0.1%SDS和EDTA。所分离样品为小鼠肝脏组织制备的不同浓度样品。根据图2对所得电泳图进行比较,专利配方(图2右)在长时间放置后,电泳至凝胶底部不会黄化(表现为溴芬兰颜色变成黄色),而对照凝胶(图2左)会在底部变黄,导致底部结果无法获得。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.

实施例3Example 3

本实例对本发明添加的备选的稳定剂苹果酸,同时添加谷氨酸和二甲基甲酰胺的聚丙烯酰胺凝胶,在配制后37摄氏度放置12天后,与当天配制的同样配方凝胶进行比较。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.

使用玻璃板或塑料板配制聚丙烯酰胺浓度12%的水溶液(T=12%,C=2.6%,N,N甲叉双丙烯酰胺浓度为1688mM)。制胶溶液中含有密度剂甘油浓度8%,并使用盐酸将pH调节至6.4-6.6之间,使用过硫酸铵和TEMED作为催化剂催化N,N甲叉双丙烯酰胺聚合交联形成三维网状的聚丙烯酰胺凝胶。对照溶液配制相同浓度的聚丙烯酰胺凝胶(催化部分为常规方法或本领域公知技术,本发明对此不进行特殊限定)。具体配方如表3所示: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:

表3:实施例3的配方Table 3: Formulation of Example 3

Figure BDA0003614984960000071
Figure BDA0003614984960000071

使用Mini-Protean型电泳槽电泳,测试电泳缓冲液使用配方为50mM三羟甲基氨基甲烷、50mM 3-吗啉丙磺酸、0.1%SDS和EDTA。所分离样品为小鼠肝脏组织制备的不同浓度样品、预染marker、非预染marker和大肠杆菌裂解物。由图3所示对所得电泳图进行比较,图3中左侧凝胶图为当天配制凝胶,右侧为37摄氏度放置12天后结果,除部分条带稍有弥散外,没有明显变化。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.

实施例4Example 4

本实施例也是热加速试验,研究本发明范围内使用不同组合和浓度的缓冲剂和稳定剂对聚丙烯酰胺凝胶稳定性,尤其是条带迁移率的影响。按照下表内凝胶配方,使用玻璃板或塑料板配制聚丙烯酰胺浓度10%的水溶液(T=12%,C=2.6%),N,N甲叉双丙烯酰胺浓度为1407mM。制胶溶液中含有密度剂甘油浓度8%,并使用盐酸将pH调节至6.4-6.6之间,使用过硫酸铵和TEMED作为催化剂催化N,N甲叉双丙烯酰胺聚合交联形成三维网状的聚丙烯酰胺凝胶。对照溶液配制相同浓度的聚丙烯酰胺凝胶(催化部分为常规方法或本领域公知技术,本发明对此不进行特殊限定)。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).

配方名称recipe name 区别组分distinguishing components 配方ARecipe A 200Mm Bis-Tris;30Mm谷氨酸;10mM二甲基甲酰胺200Mm Bis-Tris; 30Mm Glutamate; 10mM Dimethylformamide 配方BRecipe B 300Mm Tris;30Mm谷氨酸;10mM二甲基甲酰胺300Mm Tris; 30Mm Glutamate; 10mM DMF 配方CRecipe C 300Mm Tris;30Mm谷氨酸;50mM二甲基甲酰胺300Mm Tris; 30Mm Glutamate; 50mM DMF

使用Mini-Protean型电泳槽电泳,测试电泳缓冲液使用配方为50mM三羟甲基氨基甲烷、50mM 3-吗啉丙磺酸、0.1%SDS和EDTA。所分离样品为非预染蛋白分子量标准,上述配方在配制后37摄氏度放置12天,和在使用当天配制凝胶比较非预染蛋白分子量标准在不同配方胶内和不同保存时间的胶内的迁移位置,蛋白分子量迁移率公式为:lgMr=-b*mR+K。因凝胶分离范围为线性,但一个浓度只针对一定的蛋白分子量分离范围内是线性关系,例如10%浓度凝胶,在蛋白分子质量40-70KDa范围内呈线性关系。结果如图4-9所示,不同组分配比和保存时间,蛋白marker迁移位置基本一致。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.

对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。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.

此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。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.
CN202210440812.2A 2022-04-25 2022-04-25 Gel for separating protein through electrophoresis, buffer solution used in cooperation with gel and kit of gel Pending CN114791458A (en)

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