CN115508563A

CN115508563A - Protein membrane transferring liquid and preparation method and application thereof

Info

Publication number: CN115508563A
Application number: CN202211158536.7A
Authority: CN
Inventors: 丁文鹏; 王靖鹏; 丁闻; 滕彩英
Original assignee: Shenzhen Kuyuan Biotechnology Co ltd
Current assignee: Shenzhen Kuyuan Biotechnology Co ltd
Priority date: 2022-09-22
Filing date: 2022-09-22
Publication date: 2022-12-23

Abstract

The invention discloses a protein membrane-transferring liquid and a preparation method and application thereof, belonging to the technical field of biological immunology. The protein membrane-transferring liquid comprises a pH buffer pair, tris (hydroxymethyl) methylglycine, a complexing agent, a multifunctional additive and ultrapure water, wherein the pH of the protein membrane-transferring liquid is 8.3-8.4. The raw materials of the protein membrane-transferring liquid are easy to purchase, the protein membrane-transferring liquid is simple to prepare, the result after membrane transferring is reliable, the developing result is stable, reliable and credible compared with the developing result of the traditional membrane-transferring liquid, crystallization is not easy to form after long-term use, the electric membrane transferring time is obviously reduced, the membrane transferring can be completed in 20min under the current of 400mA, and the immune protein marking efficiency is obviously improved. And the protein transmembrane liquid does not contain volatile solution methanol which is harmful to human bodies.

Description

Protein membrane transferring liquid and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological immunology, and relates to a protein membrane transferring solution for protein membrane transfer between an electrophoresis gel and a solid-phase carrier membrane in an immune protein imprinting technology, and a preparation method and application thereof.

Background

Transmembrane is a common technique used in many research and diagnostic laboratories. Western Blot (immunoblotting) protein samples separated by PAGE after electrophoresis on polyacrylamide gels are transferred to a solid support (e.g., nitrocellulose membrane) which adsorbs the proteins in a non-covalent manner and retains the type of electrophoretically separated polypeptides and their biological activity. PVDF membranes are charged after methanol activation, and thus will adsorb proteins when the membrane is transferred. The hybridization membranes used for Western blot are mainly of two types: NC membrane (nitrocellulose film) and PVDF membrane (polyvinylidene fluoride film) can be selected according to the binding capacity of target protein and membrane and the aperture of membrane.

The film transfer method is divided into a wet transfer method and a semi-dry transfer method, and the two methods have respective advantages and disadvantages. Wet transfer membranes are most efficient, but waste reagents and solutions and are time consuming; the semi-dry transfer saves time and reagents and is more suitable for transferring proteins with smaller molecular weight. The conventional wet transfer process requires a significant amount of time, typically over one-half hour, to formulate the reagents and transfer process. And in this process because the effect of electric current can produce a large amount of heats, and the high temperature can reduce and change membrane efficiency, so still need a continuous low temperature environment to increase and change membrane efficiency, need add ice-cube and ice bag auxiliary cooling around changeing the membrane appearance, increaseed experimental cost and increased whole immune protein seal time.

After recognizing the problems, the inventor researches and provides a rapid film transferring liquid, and tests through multiple experiments prove that the film transferring efficiency is obviously improved while the film transferring result is credible, and the rapid film transferring liquid can completely replace the existing common film transferring liquid.

Disclosure of Invention

The first purpose of the invention is to provide a protein membrane transferring liquid, which is applied to an immune protein imprinting technology for protein membrane transferring, solves the problem that a large amount of time is consumed in the transfer process by using a traditional wet transfer method in the prior art, obviously improves the membrane transferring efficiency, and can ensure the reliability of the membrane transferring result.

The second purpose of the invention is to provide a preparation method of the protein transmembrane fluid.

The third purpose of the invention is to provide the application of the protein-transferring liquid in the protein transferring between an electrophoresis gel (SDS-PAGE gel) and a solid-phase carrier membrane (PVDF membrane, NC membrane and nylon membrane) in the immune protein imprinting technology.

In order to solve the technical problems, the invention adopts the following technical scheme:

a protein transmembrane fluid comprising: pH buffer pair, tricine, complexing agent, multifunctional additive and ultrapure water, wherein the pH of the protein membrane transferring liquid is 8.3-8.4, and the multifunctional additive plays roles of corrosion prevention, oxidation resistance and bacteria inhibition.

Tris (hydroxymethyl) methylglycine (Tricine) is a commonly used organic compound for buffer solutions, which has a lower negative charge than glycine and allows faster migration, while its high ionic strength results in more ionic and less protein movement. The content of the tri (hydroxymethyl) methyl glycine is 130mM-150mM.

The pH buffer pair is preferably a buffer pair consisting of 4-hydroxyethyl piperazine propanesulfonic acid (HEPPS), 3- (N-morpholinyl) propanesulfonic acid (MOPS) or Tris and glycine. More preferably a buffer pair of Tris and glycine. The content of the trihydroxymethyl aminomethane is 330mM-340mM, and the content of the glycine is 250mM-270mM. The buffer pair has the function of keeping the pH value of the adjusted membrane transferring liquid stable.

The complexing agent is selected from disodium ethylenediaminetetraacetic acid (EDTA-2 Na) or ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) to eliminate the effect of some impurity ions (such as calcium and potassium ions) on the rotating membrane. Preferably disodium ethylenediaminetetraacetate (EDTA-2 Na) in an amount of 2mM-3mM.

The multifunctional auxiliary agent is at least one selected from sodium bisulfite, sodium sulfite, metabisulfite (such as potassium metabisulfite and sodium metabisulfite), and sodium hyposulfite. Sodium bisulfite is preferred, in an amount of 1mM to 2mM.

As a preferred embodiment of the present invention, the protein transmembrane fluid comprises: 336mM Tris, 260mM glycine, 140mM Tris (hydroxymethyl) methylglycine, 2.5mM disodium EDTA, 1.3mM sodium bisulfite.

The preparation method of the protein membrane-transferring liquid comprises the following steps:

taking a 1L beaker containing 500ml of ultrapure water, adding tris (hydroxymethyl) aminomethane, glycine and NaHSO ₃ Tris (hydroxymethyl) methylglycine and EDTA-2Na were dissolved by stirring thoroughly. Ultrapure water was replenished to approximately 1000ml and the pH adjusted to 8.3-8.4 using HCl or NaOH. The volumetric flask has a constant volume of 1L.

In view of the excellent performance of the protein transfection solution of the present invention in the immune western blotting technique, the membrane transfer method of the protein transfection solution of the present invention is as follows:

a: starting to prepare filter paper, sponge and a solid phase carrier membrane (such as PVDF membrane) required by membrane transfer before electrophoresis is finished;

b: soaking the solid phase carrier membrane in anhydrous methanol for 30s before use to activate the positive charge group on the solid phase carrier membrane, so that the solid phase carrier membrane can be combined with negatively charged protein more easily;

c: after electrophoresis, the glass plate is pried open, and the gel is scraped off gently to avoid scraping the gel.

D: flatly placing a base of the transfer tank, flatly laying and stacking a sponge I, a filter paper I soaked with the protein transfer solution, a solid-phase carrier membrane, a stripped gel, a filter paper II soaked with the protein transfer solution and a sponge II on a graphite electrode in sequence, and performing pressing and bubble removal treatment on each layer in the stacking process.

E: sucking the redundant protein membrane-transferring liquid on and around the lamination layer by using clean gauze, finally buckling the upper cover of the transfer groove, adding the protein membrane-transferring liquid into the membrane-transferring groove, switching on a power supply by using a biorad electrophoresis apparatus, setting a constant current of 400mA, and starting membrane transfer.

Compared with the prior art, the invention has the beneficial effects that:

the protein transfer solution provided by the invention has the advantages that the raw materials are easy to purchase, the preparation of the protein transfer solution is simple, the result after the membrane transfer is reliable, the developing result is more stable and reliable compared with the developing result of the traditional transfer solution, the crystallization is not easy to form after long-term use, the electric transfer time is obviously reduced, the membrane transfer can be completed only in 20min under the heavy current of 400mA, the immune protein imprinting efficiency is obviously improved, and the heat generation in the membrane transfer process is less. And the protein membrane transferring liquid does not contain volatile solution methanol harmful to human bodies.

Drawings

FIG. 1 is a diagram showing the effect of the protein membrane-transecting reagent of the present invention.

Detailed Description

In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the attached drawings.

The invention relates to a protein membrane-transforming liquid, which comprises: pH buffer pair, tricine, complexing agent, multifunctional additive and ultrapure water, wherein the pH of the protein membrane transferring liquid is 8.3-8.4, and the multifunctional additive plays roles of corrosion prevention, oxidation resistance and bacteria inhibition.

Tris (hydroxymethyl) methylglycine (Tricine) is a commonly used organic compound for buffer solutions, which has a lower negative charge than glycine and allows faster migration, while its high ionic strength results in more ionic and less protein movement. The content of the tri (hydroxymethyl) methylglycine is 130mM-150mM.

The pH buffer pair is preferably a buffer pair consisting of 4-hydroxyethyl piperazine propanesulfonic acid (HEPPS), 3- (N-morpholinyl) propanesulfonic acid (MOPS) or Tris and glycine. More preferably a buffer pair of Tris and glycine. The content of the trihydroxymethyl aminomethane is 330mM-340mM, and the content of the glycine is 250mM-270mM.

The multifunctional adjuvant is at least one selected from sodium bisulfite, sodium sulfite, metabisulfite (such as potassium metabisulfite and sodium metabisulfite), and sodium hyposulfite. Sodium bisulfite is preferred, in an amount of 1mM to 2mM.

As a preferred embodiment of the present invention, an eggThe white transfer membrane liquid comprises: 336mM Tris, 260mM glycine, 140mM Tris (hydroxymethyl) methylglycine, 2.5mM disodium EDTA, 1.3mM sodium bisulfite. The process for preparing the protein transmembrane liquid comprises the following steps: A1L beaker containing 500ml of ultrapure water was charged with (336 mM) tris (hydroxymethyl) aminomethane 40.703g, (260 mM) glycine 19.5182g, (1.3 mM) NaHSO ₃ 0.135g (140 mM) of tris (hydroxymethyl) methylglycine 25.08394g and (2.5 mM) of EDTA-2Na 0.841g, and sufficiently stirred to dissolve. Ultrapure water was supplemented to approximately 1000ml and the pH adjusted to 8.3-8.4 using HCl or NaOH. The volumetric flask has a constant volume of 1L.

And (3) film transferring:

the materials required for film transfer begin to be prepared 20min before electrophoresis is finished. For one film, 2 filter papers and 2 sponges (the length is generally 8.1-8.3 cm, the width is actually measured according to the size of the cut glue, but the glue generally shrinks, so the film can be cut by 8 cm) and 1 PVDF film are prepared. The filter paper and membrane must be cut with clean gloves because the proteins on the hands contaminate the membrane. Before the PVDF membrane is used, the PVDF membrane is soaked in anhydrous methanol for 30s to activate positive charge groups on the PVDF membrane, so that the PVDF membrane can be more easily combined with negatively charged proteins, and methanol is added into a membrane transferring liquid when small molecular proteins are transferred.

After the electrophoresis, shell the glue after prizing the glass board, the action will be light when prizing the glass board to prize in two edges of glass board gently repeatedly, take away the glass board after the glass board begins to become flexible, then scrape the concentrated gel gently, will avoid scraping the gel of separation brokenly. After taking out the gel, the upper and lower parts should be clearly distinguished, and a small corner can be cut at the upper right corner of the gel to be used as a mark.

Putting on gloves, flatly placing a base of the transfer tank, flatly laying and stacking a sponge I, a filter paper I soaked with the protein transfer solution, a PVDF membrane (after laying, a corner can be cut at the right upper corner of the PVDF membrane to conveniently identify the protein surface and the non-protein surface after membrane transfer), the stripped gel, a filter paper II soaked with the protein transfer solution and a sponge II on the graphite electrode in sequence, and carrying out pressure bubble removal treatment on each layer in the stacking process. The specific operation can be to remove air bubbles by using a gun head, or a pipette, or a glass rod or a cylindrical test tube, etc., and the thickness of one air bubble has great influence on the result, so the air bubbles should be removed.

Sucking up the redundant protein membrane transferring liquid on and around the lamination layer by using clean gauze, finally buckling the upper cover of the transferring groove, adding the protein membrane transferring liquid into the transferring groove, switching on a power supply by using a biorad electrophoresis apparatus, setting a constant current of 400mA, and starting to transfer the membrane.

Results of the experiment

After the membrane is rotated by constant current of 400mA for 20 minutes, a piece of membrane with protein being rotated well and a piece of hollow gel can be obtained. The results are shown in FIG. 1 (membrane on the left of the figure, gel after membrane rotation on the right of the figure). Therefore, the protein transfer solution can transfer the protein to the membrane in a very short time, and the transfer result of the protein after membrane transfer is reliable.

Since the principle is unchanged, the same effect can be achieved when the kind of glue and film is replaced.

The protein transmembrane liquid has no requirement on the molecular weight of the protein, and the transmembrane time is properly increased if the protein needing transmembrane is large. The above experiment used a maker, which contained 10-170kd of all proteins, and the membrane transfer was successful within the membrane transfer time.

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.

Claims

1. A protein membrane transfer solution, comprising: pH buffer pair, tris (hydroxymethyl) methylglycine, complexing agent, multifunctional additive and ultrapure water, wherein the pH of the protein membrane transferring liquid is 8.3-8.4;

the pH buffer pair is selected from a buffer pair consisting of 4-hydroxyethyl piperazine propanesulfonic acid, 3- (N-morpholinyl) propanesulfonic acid or trihydroxymethyl aminomethane and glycine;

the complexing agent is selected from disodium ethylene diamine tetraacetate or ethylene glycol bis (2-aminoethyl ether) tetraacetic acid;

the multifunctional auxiliary agent is at least one of sodium bisulfite, sodium sulfite, metabisulfite and sodium hyposulfite.

2. The protein transmembrane solution according to claim 1, wherein the pH buffer pair is a buffer pair of tris and glycine; the content of the trihydroxymethyl aminomethane is 330mM-340mM, and the content of the glycine is 250mM-270mM.

3. The protein transmembrane solution according to claim 1, wherein the tris (hydroxymethyl) methylglycine is present in an amount of 130mM to 150mM.

4. The protein membrane transfer solution of claim 1, wherein the complexing agent is disodium ethylenediaminetetraacetate in an amount of 2mM to 3mM.

5. The protein transfection solution of claim 1, wherein the multifunctional auxiliary agent is sodium bisulfite and is present in an amount of 0.1mM to 0.2mM.

6. A protein membrane transfer solution, comprising: 336mM Tris, 260mM glycine, 140mM Tris (hydroxymethyl) methylglycine, 2.5mM disodium EDTA, 1.3mM sodium bisulfite.

7. A method of preparing a protein transfection solution according to any one of claims 1 to 6, comprising the steps of:

adding the raw materials into ultrapure water, fully stirring and dissolving, supplementing ultrapure water to be close to 1000ml, and adjusting the pH value to 8.3-8.4 by using HCl or NaOH; the volumetric flask has a constant volume of 1L.

8. Use of a protein-transmembrane fluid according to any one of claims 1 to 6 as a protein-transmembrane fluid in immunowestern blotting.

9. The use according to claim 6, wherein the protein-transmembrane solution is subjected to protein-transmembrane reaction between an electrophoresis gel and a PVDF membrane or an NC membrane or a nylon membrane as a solid-phase carrier.

10. Use according to claim 9, characterized in that it comprises the following steps:

a: preparing filter paper, sponge and a solid phase carrier membrane required by membrane transfer before electrophoresis is finished;

b: soaking the solid phase carrier membrane in anhydrous methanol before use;

c: after electrophoresis is finished, prying the glass plate open, and slightly scraping the gel;

d: flatly placing a base of the transfer tank, flatly laying and stacking a sponge I, a filter paper I soaked with the protein transfer solution, a solid-phase carrier membrane, a stripped gel, a filter paper II soaked with the protein transfer solution and a sponge II on a graphite electrode in sequence, and performing pressure bubble removal treatment on each layer in the stacking process;

e: sucking the redundant protein membrane transferring liquid on and around the lamination layer by using clean gauze, finally buckling the upper cover of the transferring groove, adding the protein membrane transferring liquid into the transferring groove, switching on a power supply by using a biorad electrophoresis apparatus, setting a constant current of 400mA, and starting membrane transferring.