阅读说明：本技术 一种用于小分子量蛋白质分离的聚丙烯酰胺凝胶及其制备方法 (A kind of polyacrylamide gel and preparation method thereof for small molecular weight protein separation ) 是由 杨颂 王绪德 张高英 于 2019-09-16 设计创作，主要内容包括：本发明涉及蛋白质聚丙烯酰胺凝胶电泳技术领域,尤其是一种用于小分子量蛋白质分离的聚丙烯酰胺凝胶,该凝胶包括丙烯酰胺、N,N-甲叉双丙烯酰胺、超纯水、凝胶缓冲液、甘油、过硫酸铵溶液和四甲基乙二胺,凝胶缓冲液包括硼酸和Tris-HCl溶液,凝胶缓冲液的pH值为7.0。本发明以Tris-硼酸为缓冲体系制备聚丙烯酰胺凝胶,对小分子蛋白质实现很好的分离,蛋白条带清晰锐利,分离效果与目前常用的小分子蛋白分离凝胶Tris-Tricine聚丙烯酰胺凝胶相当,分离效果甚至优于Tris-Tricine,这种方式大大降低了试剂的使用成本,并且可得到更优的分离效果。(The present invention relates to protein technique of polyacrylamide gel electrophoresis fields, especially a kind of polyacrylamide gel for small molecular weight protein separation, the gel includes acrylamide, N, N- methylene diacrylamide, ultrapure water, gel buffer liquid, glycerol, ammonium persulfate solution and tetramethylethylenediamine, gel buffer liquid includes boric acid and Tris-HCl solution, and the pH value of gel buffer liquid is 7.0.The present invention prepares polyacrylamide gel using Tris- boric acid as buffer system, separation well is realized to small protein, protein band is clearly sharp keen, separating effect is suitable with currently used small molecular protein separating gel Tris-Tricine polyacrylamide gel, separating effect is even better than Tris-Tricine, this mode greatly reduces the use cost of reagent, and available more preferably separating effect.)

1. a kind of polyacrylamide gel for small molecular weight protein separation, which is characterized in that the gel includes acryloyl Amine, N, N- methylene diacrylamide, ultrapure water, gel buffer liquid, glycerol, ammonium persulfate solution and tetramethylethylenediamine are described Gel buffer liquid includes boric acid and Tris-HCl solution.

2. a kind of polyacrylamide gel for small molecular weight protein separation according to claim 1, feature exist In the pH value of the gel buffer liquid is 7.0.

3. a kind of polyacrylamide gel for small molecular weight protein separation according to claim 2, feature exist In the concentration of the Tris is 50-200mM, and the concentration of the boric acid is 50-200mM.

4. a kind of its preparation of the polyacrylamide gel according to claim 1 to 3 for small molecular weight protein separation Method, which comprises the steps of:

Step 1: preparing 30% acrylamide stock solution, wherein acrylamide and N, the mass ratio of N- methylene diacrylamide are 29:1, specific implementation steps are as follows:

A, firstly, weighing in the acrylamide of 29g and the N of 1g, N- methylene diacrylamide addition mixing vessel；

B, ultrapure water then, is added into container, so that acrylamide and N, N- methylene diacrylamide is dissolved in ultrapure water；

C, finally, mixed solution is settled to 100mL；

Step 2: prepare 10% ammonium persulfate stock solution, weigh 100mg ammonium persulfate be dissolved in 1mL ultrapure water obtain mixing it is molten Liquid；

Step 3: 2.5 × gel buffer liquid is prepared, specific implementation steps are as follows:

A, it weighs the mixing of 3.7856g Tris base and 2.3175g boric acid to be added to the container, and 80mL ultrapure water is added；

B, continuously adding HCl into mixed solution and being titrated to pH is 7.0；

C, mixed solution is finally settled to 100mL；

Step 4: preparing 50% glycerol, weigh 50g glycerol, 50mL ultrapure water is added and obtains mixed solution；

Step 5: 5% concentration glue is prepared, specific implementation steps are as follows:

(1), firstly, taking 30% acrylamide stock solution in 1mL step 1, the then gel buffer liquid in 2.4mL step 3；

(2), 10% ammonium persulfate stock solution in 30 μ L steps 2 then, is added, and ultrapure water is added and is settled to 6mL；

(3), finally, the tetramethylethylenediamine of 6 μ L is added before facing encapsulating；

Step 6: 10% separation gel is prepared, specific implementation steps are as follows:

S1,30% acrylamide stock solution in 4mL step 1 is taken, adds 2.5 × gel buffer liquid in 4.8mL step 3, Continuously add 50% glycerol in 1.2mL step 4；

S2, be added 60 μ L steps 2 in 10% ammonium persulfate stock solution, then plus ultrapure water be settled to 12mL；

S3, face before encapsulating the tetramethylethylenediamine that 6 μ L are added.

Technical field

The present invention relates to protein technique of polyacrylamide gel electrophoresis fields, more particularly to one kind to be used for small-molecular-weight egg The polyacrylamide gel and preparation method thereof of white matter separation.

Background technique

Polyacrylamide gel electrophoresis is that a kind of analyzed using polyacrylamide gel for medium progress Separation of Proteins is examined The technology of survey, polyacrylamide gel are the macromolecular being polymerized by acrylamide and N, N- methylene diacrylamide, amide The effect that reticular structure has molecular sieve is formed after side chain polymerization, the netted pore size and gel strength of gel-forming are at anti- Than concentration is bigger, and aperture is smaller, and separation small molecular weight protein ability is stronger；Conversely, concentration is smaller, aperture is bigger, and separation is big Molecule protein effect is preferable, protein in polyacrylamide gel when electrophoresis, its mobility depend on three it is main because Element: the size of gel porosity, its institute's band net charge and bulk of molecule and shape are influenced when eliminating the influence of charge Mobility is exactly molecular size range and porosity, and the principle of gel electrophoresis technology protein isolate is based on this；

There are many kinds of the buffer systems for being used to prepare polyacrylamide gel, such as Tris-Glycine, Bis Tris- MOPS, Tris-Acetate, Tris-Tricine etc..Wherein most contributes to separate the medium and above sized molecules amount Protein needs the gel of very high concentration for small molecular weight protein separation, and the brittleness of the high gel of concentration is high, for possible There are certain difficulties for subsequent recovery albumen.Under same concentrations gelation condition, to the buffer system of small molecular weight protein separation Common Tris-Tricine, but the cost is relatively high for Tricine reagent.

Summary of the invention

The purpose of the present invention is to solve the buffer reagent costs that small molecular weight protein separation exists in the prior art High disadvantage, and a kind of polyacrylamide gel and preparation method thereof for small molecular weight protein separation proposed.

To achieve the goals above, present invention employs following technical solutions:

A kind of polyacrylamide gel for small molecular weight protein separation is designed, which includes acrylamide, N, N- methylene diacrylamide, ultrapure water, gel buffer liquid, glycerol, ammonium persulfate solution and tetramethylethylenediamine, the gel are slow Fliud flushing includes boric acid and Tris-HCl solution.

Preferably, the pH value of the gel buffer liquid is 7.0.

Preferably, the concentration of the Tris is 50-200mM, and the concentration of the boric acid is 50-200mM.

The present invention also provides it is a kind of for small molecular weight protein separation polyacrylamide gel preparation method, Include the following steps:

Step 1: preparing 30% acrylamide stock solution, wherein acrylamide and N, the mass ratio of N- methylene diacrylamide For 29:1, specific implementation steps are as follows:

A, firstly, weighing in the acrylamide of 29g and the N of 1g, N- methylene diacrylamide addition mixing vessel；

B, ultrapure water then, is added into container, so that acrylamide and N, N- methylene diacrylamide is dissolved in ultrapure water In；

C, finally, mixed solution is settled to 100mL；

Step 2: preparing 10% ammonium persulfate stock solution, weigh 100mg ammonium persulfate and be dissolved in 1mL ultrapure water and mixed Solution；

Step 3: 2.5 × gel buffer liquid is prepared, specific implementation steps are as follows:

A, it weighs the mixing of 3.7856g Tris base and 2.3175g boric acid to be added to the container, and 80mL ultrapure water is added；

B, continuously adding HCl into mixed solution and being titrated to pH is 7.0；

C, mixed solution is finally settled to 100mL；

Step 4: preparing 50% glycerol, weigh 50g glycerol, 50mL ultrapure water is added and obtains mixed solution；

Step 5: 5% concentration glue is prepared, specific implementation steps are as follows:

(1), firstly, taking 30% acrylamide stock solution in 1mL step 1, the then gel buffer in 2.4mL step 3 Liquid；

(2), 10% ammonium persulfate stock solution in 30 μ L steps 2 then, is added, and ultrapure water is added and is settled to 6mL；

(3), finally, the tetramethylethylenediamine of 6 μ L is added before facing encapsulating；

Step 6: 10% separation gel is prepared, specific implementation steps are as follows:

S1,30% acrylamide stock solution in 4mL step 1 is taken, adds 2.5 × gel buffer in 4.8mL step 3 Liquid continuously adds 50% glycerol in 1.2mL step 4；

S2, be added 60 μ L steps 2 in 10% ammonium persulfate stock solution, then plus ultrapure water be settled to 12mL；

S3, face before encapsulating the tetramethylethylenediamine that 6 μ L are added.It is proposed by the present invention a kind of for small molecular weight protein point From polyacrylamide gel and preparation method thereof, beneficial effect is: the present invention prepares poly- using Tris- boric acid as buffer system Acrylamide gel, to small protein realization separation well, protein band is clearly sharp keen, and separating effect is commonly used with current Small molecular protein separating gel Tris-Tricine polyacrylamide gel it is suitable；Tricine is replaced by boric acid to coagulate Glue preparation cost substantially reduces and does not influence the separating effect to small protein, while polyacrylamide prepared by the present invention Gel is suitable for general Tris-Glycine electrophoretic buffer, and polyacrylamide gel prepared by the present invention is neutral system, The gel of preparation can long-term preservation do not hydrolyze, do not change gel resolution ratio, future can be used to prepare precast gel, and this mode is big The use cost of reagent is reduced greatly.

Detailed description of the invention

Fig. 1 is prepared by a kind of method of polyacrylamide gel for small molecular weight protein separation electrophoresis of the invention Separation gel be 10% Tris-Tricine gel and Tris- boric acid gel protein isolate comparison diagram；

Fig. 2 is prepared by a kind of method of polyacrylamide gel for small molecular weight protein separation electrophoresis of the invention Separation gel be 18% Tris- boric acid gel and 18% Tris-Tricine gel protein isolate Marker comparison diagram.

Specific embodiment

The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation Example is only a part of the embodiment of the present invention, instead of all the embodiments.