WO2020221182A1

WO2020221182A1 - 05sar-page, preparation method therefor, and application thereof

Info

Publication number: WO2020221182A1
Application number: PCT/CN2020/087126
Authority: WO
Inventors: 黄丽群; 姜凌; 李从刚
Original assignee: 中国科学院精密测量科学与技术创新研究院
Priority date: 2019-04-29
Filing date: 2020-04-27
Publication date: 2020-11-05
Also published as: CN110133091A; CN110133091B

Abstract

Provided are a 05SAR-PAGE, a preparation method therefor, and an application thereof, relating to a gel electrophoresis separation technique. The 05SAR-PAGE is a gel prepared from sodium lauroyl sarcosinate with a mass-volume ratio of 0.05%, and comprises a lower separation gel and an upper concentration gel. The lengths and thicknesses of the lower separation gel and the upper concentration gel are the same, and the ratio of the height of the lower separation gel to the height of the upper concentration gel is equal to 2:1 to 8:1. The present invention resolves the shortcomings in which SDS-PAGE cannot be used to analyze physiological state protein modification and native-PAGE cannot use protein markers for calibration and cannot be directly applied to the analysis of acidic and basic proteins. The present invention has universality, simple operation, and low costs, and is widely applicable to fundamental and applied research in the fields of molecular dynamics, enzymology, medicine, etc., bringing extensive convenience to the scientific analysis of proteins for experimenters.

Description

05SAR-PAGE and its preparation method and application

Technical field

The invention relates to a gel electrophoresis separation technology, in particular to a 05SAR-PAGE and its preparation method and application.

Background technique

Protein is an important component of living organisms and the main bearer of life activities. The biological functions of proteins often exist in a polymerized state, a complex state or a modified state. At present, it is still challenging to accurately identify different states of proteins and protein interactions.

SDS-PAGE and Native-PAGE are currently the most commonly used protein identification methods in the field of protein research. SDS is a strong anionic detergent that can bind to proteins and break the structure of the globe-like protein. The molecular weight of the protein is related to the amount of charge carried by the protein. Therefore, the migration speed of the protein during electrophoresis depends only on the molecular weight of the protein. , And can use protein Marker to indicate the molecular weight of the target protein. Because SDS-PAGE destroys the natural conformation of the protein, it is often used in the study of denatured proteins, so the modified state or complex state of the protein is hardly observed in SDS-PAGE.

Native-PAGE is a non-denaturing gel electrophoresis without SDS. During the electrophoresis process, the protein can remain intact. Because the migration rate of the protein in Native-PAGE is affected by the molecular weight, protein shape and charge, Therefore, Native-PAGE does not have a unified protein Marker calibration, which causes problems for analyzing the molecular weight, polymer state and complex state of the protein. At the same time, due to the different charges of acidic protein and basic protein, it brings inconvenience to the experimental operation.

SAR-PAGE is a gel made of sodium lauroyl sarcosinate (Sarkosyl, SAR) instead of SDS. SAR is a mild anionic detergent, because it only binds to protein and does not bind to polyethylene. Alcohol binding, therefore, SAR-PAGE of 0.1% w/v SAR has been used in immunological studies of PEGylated proteins in recent years. Experiments have proved that 0.1% w/v SAR will destroy the structure of some conventional proteins, so SAR-PAGE containing 0.1% w/v SAR is not used for protein aggregation state and protein enzyme activity analysis.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the traditional protein gel electrophoresis technology, and provide a 05SAR-PAGE and its preparation method and application; the present invention does not destroy the secondary structure of the protein, and can determine the target protein of electrophoresis at the same time. Standard, semi-quantitative analysis, simple operation, low cost, suitable for the most commonly used Tris-Glycine electrophoresis system.

The purpose of the present invention is achieved as follows:

The invention includes a gel electrophoresis system containing 0.05% w/v SAR and a protein sample solution of 0.1-0.5 mM.

The 0.05% SAR gel electrophoresis system includes a loading buffer containing 05SAR-PAGE, a loading buffer containing 0.05% w/v SAR, a running buffer containing 0.05% w/v SAR, and a protein marker containing 0.05% w/v SAR.

1. 05SAR-PAGE

Protein separation gel prepared with 0.05% mass volume ratio (w/v) sodium lauroyl sarcosinate (abbreviated as 05SAR-PAGE)

Including lower separation glue and upper concentrated glue:

The length and thickness of the lower separation glue and the upper concentrated glue are the same;

Lower separation glue height: upper concentrated glue height=2:1 to 8:1;

According to the molecular weight of the target protein, prepare the lower separation gel with different concentrations of acrylamide; the lower separation gel is composed of 5 substances, including 30% acrylamide solution, 1.5mol/L Tris pH8.8, 10%w/v ammonium persulfate , 5% w/v SAR and tetramethylethylenediamine (TEMED), the lower layer separation gel formula includes 5 kinds: 6% acrylamide gel formula (Table 1), 8% acrylamide gel formula (Table 2) , 10% acrylamide gel formula (table 3), 12% acrylamide gel formula (table 4), 15% acrylamide gel formula (table 5);

The formula of the upper separation gel includes one: 5% acrylamide gel (Table 6).

2. Preparation method of 05SAR-PAGE

This method includes the following steps:

① Preparation of gel plate

A. Preparation of separating glue

a. Prepare the separating gel according to the amount in the table, and add distilled water, 30% acrylamide solution, 1.5mol/L Tris, pH8.8, 10%w/v ammonium persulfate, 5%w/v in a clean small beaker. SAR, mix gently, finally add tetramethylethylenediamine (TEMED), mix gently again;

b. Use a 1mL pipette to mix the above mixed solution and add it to the gap between the long and short rubber plates. Separate the glue 53-63mm high. Use a 1mL syringe to take a little distilled water and fill the water seal along the long glass plate. After 15-30min, Use filter paper to absorb excess water;

B. Preparation of concentrated glue

a. Prepare the concentrated glue according to the amount in the table, add distilled water, 30% acrylamide solution, 1.5mol/L Tris pH8.8, 10%w/v ammonium persulfate, 5%w/v SAR in a clean small beaker in turn , Mix gently, finally add tetramethylethylenediamine (TEMED), mix gently again.

b. Use a 1mL pipette to mix the above-mentioned mixture and add the upper layer of the separating gel, quickly insert the sample tank template, and leave it for 20-60 minutes. After the concentrated gel is completely solidified, take out the sample tank template and put it in a 4℃ refrigerator for later use;

②Preparation of loading buffer

A. Prepare 50mM Tris-HCl pH6.8, 0.05%w/v SAR, 0.1%w/v bromophenol blue, 10%v/v glycerin;

B. Add deionized water to dissolve and dilute to 5mL;

C. Store in small portions at room temperature after packaging;

③Preparation of running buffer:

A. Prepare 25mM Tris, 0.25mM glycine, 0.05% w/v SAR;

B. Add deionized water to dissolve and dilute to 5mL;

④ Preparation of protein Marker

The protein Marker contains 4-8 molecular weight gradients, and the protein molecular weight ranges from 5kDa to 300kDa. Dissolve the protein Marker with a concentration of 0.05-0.5mM in the loading buffer solution;

⑤ Preparation of protein samples

Mix the protein sample with a concentration of 0.05-0.5 mM with the loading buffer at a volume ratio of 1:1 to obtain the final sample.

The present invention has the following advantages and positive effects:

① In addition to the features of traditional SDS-PAGE and Native-PAGE that can separate proteins in different states, and the operation is simple and convenient, the present invention overcomes the inability of SDS-PAGE to analyze physiological state protein modification and Native-PAGE that cannot be calibrated by protein Marker. It cannot be directly used to analyze the shortcomings of acidic protein and basic protein;

②Experimental results prove that the method has universal applicability, simple operation and low cost;

③It can be widely used in basic and applied research in the fields of molecular dynamics, enzymology, medicine, etc., bringing extensive convenience to the scientific analysis of proteins for the majority of experimenters.

Description of the drawings

Figure 1 is a schematic diagram of the electrophoresis results,

A: Traditional SDS-PAGE analysis of the results of intracellular full-length PhoR electrophoresis without heating;

B: Schematic diagram of the results of 05SAR-PAGE analysis of intracellular full-length PhoR electrophoresis without heating;

The first lane is the band of the protein Marker, and the second lane is the electrophoresis band of the intracellular full-length PhoR.

Figure 2 is a schematic diagram of the results of 05SAR-PAGE electrophoresis.

A: 05SAR-PAGE electrophoresis patterns before and after cytochrome C methylation. The first lane is the protein Marker band, and the second and third lanes are before and after cytochrome C methylation. After the electropherogram;

B: 05SAR-PAGE profile of the reaction of PhoB and acetyl dilithium phosphate over time.

Figure 3 is the 1H-15N HSQC spectrum of PhoB in different detergents;

A: It is the 1H-15N HSQC spectrum of 0.2mM PhoB in the absence of SAR (gray) and 0.05% (w/v) SAR (black);

B: It is the 1H-15N HSQC spectrum of 0.2mM PhoB in the absence of SDS (gray) and 0.05% (w/v) SDS (black).

Figure 4 shows the 1H-15N HSQC spectra of PhoB at different concentrations in SAR;

A: 1H-15N HSQC spectrum of 0.01mM PhoB in the absence of SAR (gray) and 0.05% (w/v) SAR (black);

B: 1H-15N HSQC spectrum of 0.mM PhoB in the absence of SAR (gray) and 0.05% (w/v) SAR (black);

C: 1H-15N HSQC spectrum of 1mM PhoB in the absence of SAR (grey) and 0.05% (w/v) SAR (black).

Figure 5 is a 05SAR-PAGE chart of 0.05mM PhoB.

The first lane is the band of the protein Marker,

The second lane is the band of PhoB; according to the molecular weight of the protein Marker, it can be analyzed that the second band is PhoB with monomer and dimerization.

Detailed ways

The following is a detailed description in conjunction with the drawings and embodiments:

1. 05SAR-PAGE

Table 1 The solution used for preparing Tris-glycine 6% 05SAR-PAGE polyacrylamide gel electrophoresis separation gel

Table 2 The solution used to prepare Tris-glycine 8% 05SAR-PAGE polyacrylamide gel electrophoresis separation gel

Table 3 Preparation of solutions used for the preparation of Tris-glycine 10% 05SAR-PAGE polyacrylamide gel electrophoresis separation gel

Table 4 Preparation of Tris-glycine 12% 05SAR-PAGE polyacrylamide gel electrophoresis separation gel solution

Table 5 The solution used for preparing Tris-glycine 15% 05SAR-PAGE polyacrylamide gel electrophoresis separation gel

Table 6 The solution used for preparing Tris-glycine 05SAR-PAGE polyacrylamide gel electrophoresis concentrated gel

2. Application of 05SAR-PAGE

Ⅰ. Used to directly separate and determine the polymer state of the protein without being affected by protein pI. As shown in Figure 1, the predicted isoelectric point of the full-length intracellular PhoR is 8.86, and the relative molecular weight is 29kDa. Early studies have shown that the protein has a dimerization state in solution. Because its isoelectric point is greater than 7.0, conventional Native-PAGE The technical operation is cumbersome, and the traditional SDS-PAGE is a denaturing gel, which can only run out the monomer band of the PhoR protein (as shown in Figure 1A). 05SAR-PAGE can not be affected by the isoelectric point of the PhoR protein and overcome the traditional Native- The shortcomings of PAGE and SDS-PAGE, PhoR protein dimer and monomer bands were detected (Figure 1B).

Ii. It is used to observe the modification state of protein monomer or polymer. As shown in Figure 2, targeted modification of proteins is of great significance for proteins to function in organisms. Since 0.05% w/v SAR has a mild effect on the structure of proteins, 05SAR-PAGE can be used to study protein modification. In the experiment, the methylation modification of cytochrome C and the phosphorylation modification of PhoB were observed by 05SAR-PAGE. Compared with the conventional SDS-PAGE, we have seen the methylation modification of the cytochrome C dimerization state.

Iii. It is used to observe the complexes formed by different proteins; 05SAR-PAGE can be used to detect different aggregation states of proteins, indicating that 0.05% w/v SAR has little destructive power on the protein aggregation interface, so it can also be used to detect the complexes formed by different proteins Things.

Iv. Used for protein purity analysis and protein molecular weight determination; 05SAR-PAGE retains the commonality of traditional gel electrophoresis and can perform purity analysis of the target protein.

Ⅴ. It is used to replace SDS and used in western bolt experiment to detect the multimeric state of protein in the cell. For proteins with high expression levels in cells, 05SAR-PAGE can be combined with immunohybridization experiments to detect the aggregation state of proteins in cells.

3. The innovation of the present invention:

1. In the experiment, it was found that 0.05% w/v SAR can bind to protein and has a mild effect on protein structure, and it has universal applicability. As shown in Figure 3, comparing A and B, it can be seen that 0.05%w/v SAR did not destroy the secondary structure of PhoB, while 0.05%w/v SDS destroyed the secondary structure of PhoB; indicating that compared to SDS, SAR is an extremely mild detergent.

2. In the experiment, the optimal protein electrophoresis concentration of 0.05-0.5mM was determined. As shown in Figure 4, in the SAR of 0.05% w/v, when the concentration of PhoB is 0.01 mM, PhoB tends to be unstructured; when the concentration of PhoB is 0.1 mM, the structure of PhoB is relatively stable, with only a part of the chemical shift of amino acids Disturbance occurred; when the concentration of PhoB is 1mM, the structure of PhoB is more stable, and only a few amino acid chemical shifts are disturbed; experiments show that when the concentration of SAR is 0.05%w/v, the higher the concentration of PhoB, the structure The more stable.

3. This two-dimensional gel electrophoresis technology realizes for the first time that protein markers can be used to analyze different aggregation states of proteins. As shown in Figure 5, according to the molecular weight indicated by the protein Marker, the dimerization and monomer of PhoB protein are observed. In the early research, the nuclear magnetic method also found that PhoB has dimerization and monomer two-state exchange in the solution.

4. How to use 05SAR-PAGE

1) Mix the 0.05-0.5mM target protein with the loading buffer in a volume ratio of 1:1;

2) Load the protein sample into 05SAR-PAGE and run at 80V for about 15 minutes; after the sample enters the separation gel, change it to 120V; when the blue line approaches the bottom of the gel plate, stop electrophoresis and turn off the power. Take out the gel, stain the gel using traditional methods, decolorize and analyze. (The voltage of electrophoresis can be increased or decreased according to the stability of the protein).

Claims

A 05SAR-PAGE, which is characterized by:

The 05SAR-PAGE is a protein separation gel prepared with 0.05% by weight of sodium lauroyl sarcosinate, including a lower layer separation gel and an upper layer concentrated gel:

The length and thickness of the lower separation glue and the upper concentrated glue are the same;

Lower separation glue height: upper concentrated glue height=2:1 to 8:1;

According to the molecular weight of the target protein, prepare the lower separation gel with different concentrations of acrylamide; the lower separation gel is composed of 5 substances, including 30% acrylamide solution, 1.5mol/L Tris pH8.8, 10%w/v ammonium persulfate , 5% w/v SAR and tetramethylethylenediamine, the lower layer separation gel formula includes 5 kinds: 6% acrylamide gel formula-Table 1, 8% acrylamide gel formula-Table 2, 10% acrylamide Gel formula-Table 3, 12% acrylamide gel formula-Table 4, 15% acrylamide gel formula-Table 5;

The formula of the upper separation gel includes one: 5% acrylamide gel-Table 6.
The preparation method of 05SAR-PAGE according to claim 1, characterized in that it comprises the following steps:

① Preparation of gel plate

A. Preparation of separating glue

a. Prepare the separating gel according to the amount in the table, and add distilled water, 30% acrylamide solution, 1.5mol/L Tris, pH8.8, 10%w/v ammonium persulfate, 5%w/v in a clean small beaker. SAR, mix gently, finally add tetramethylethylenediamine, mix gently again;

b. Use a 1mL pipette to mix the above mixed solution and add it to the gap between the long and short rubber plates. Separate the glue 53-63mm high. Use a 1mL syringe to take a little distilled water and fill the water seal along the long glass plate. After 15-30min, Use filter paper to absorb excess water;

B. Preparation of concentrated glue

a. Prepare concentrated gel according to the amount in the table, add distilled water, 30% acrylamide solution, 1.5mol/L Tris, pH8.8, 10%w/v ammonium persulfate, 5%w/v in a clean small beaker. SAR, mix gently, finally add tetramethylethylenediamine, mix gently again;

b. Use a 1mL pipette to mix the above-mentioned mixture and add the upper layer of the separating gel, quickly insert the sample tank template, and leave it for 20-60 minutes. After the concentrated gel is completely solidified, take out the sample tank template and put it in a 4℃ refrigerator for later use;

②Preparation of loading buffer

A. Prepare 50mM Tris-HCl, pH6.8, 0.05%w/v SAR, 0.1%w/v bromophenol blue, 10%v/v glycerin;

B. Add deionized water to dissolve and dilute to 5mL;

C. Store in small portions at room temperature after packaging;

③ Preparation of electrophoresis buffer:

A. Prepare 25mM Tris, 0.25mM glycine, 0.05% w/v SAR;

B. Add deionized water to dissolve and dilute to 5mL;

④ Preparation of protein Marker

The protein Marker contains 4-8 molecular weight gradients, and the protein molecular weight ranges from 5kDa to 300kDa. Dissolve the protein Marker with a concentration of 0.05-0.5mM in the loading buffer solution;

⑤ Preparation of protein samples

Mix the protein sample with a concentration of 0.05-0.5 mM with the loading buffer at a volume ratio of 1:1 to obtain the final sample.
The application of 05SAR-PAGE according to claim 1, characterized in that:

Ⅰ. It is used to directly separate and determine the polymer state of the protein without being affected by the pI value of the protein;

Ⅱ. It is used to observe the modification state of protein monomer or polymer;

Ⅲ. Used to observe the complexes formed by different proteins;

Iv. Used for protein purity analysis and protein molecular weight determination;

Ⅴ. Instead of SDS-PAGE, it is used in western bolt experiments to see the polymorphism of proteins in cells.
A gel electrophoresis protein Marker, characterized in that the protein standard is dissolved in a loading buffer solution with a concentration of 0.05-0.5mM, and the loading buffer contains 0.05% w/v SAR.
The gel electrophoresis protein Marker of claim 4, wherein the loading buffer contains 50 mM Tris-HCl, pH 6.8, 0.1% w/v bromophenol blue, 10 %V/v glycerol.
The gel electrophoresis protein Marker of claim 4, wherein the protein standard contains 4-8 molecular weight gradients, and the protein molecular weight ranges from 5kDa to 300kDa.