KR20110058957A - Transfer membranes for western blot integrated with papers and process for manufacturing the same - Google Patents

Abstract

PURPOSE: An integrated membrane for western blot and a method for manufacturing the same are provided to increase the sensitivity of protein detecting and diagnosing processes. CONSTITUTION: A method for manufacturing an integrated membrane for western blot includes the following: A spinning solution is obtained by dissolving a hydrophobic polymer material in a solvent. Nano fiber web based on hydrophobic polymer is obtained by spinning the spinning solution. The nano fiber web is laminated to obtain a membrane for western blot. Paper for the western blot, on which an adhesive is applied, is laminated with the membrane in order to obtain an integrated membrane for the western blot.

Description

Transfer Membranes for Western Blot Integrated with Papers and Process for Manufacturing the Same}

The present invention relates to an integrated membrane for western blot and a method for manufacturing the same, and specifically, a transfer membrane for a western blot in which a nanofiber layer and a paper layer formed by electrospinning are integrally formed, and a manufacture thereof. It is about a method.

Western blot is a technique for finding a specific protein from several protein mixtures. It uses an antibody to the protein you are looking for to generate an antigen-antibody reaction to determine the presence of a specific protein. One of the most effective ways to find out.

The transfer membrane used in the western blot is made of a porous membrane made of a material such as nitrocellulose, polyvinylidene fluoride (PVdF), or nylon (Nylon), which has a hydrophobic interaction with a protein. come. Such membranes are manufactured by methods such as dry, wet, and dry-wet casting by phase separation, in which a solvent and a polymer are poured into a non-solvent such as water. The unit price is high and there is a limit to mass production.

Currently, Western blot is used to cut PVdF membranes to gel size and soak them in 100% methanol for 1-5 minutes to enable hydrophobic interaction between proteins and gels in gels. . Carefully place the activated PVdF membrane on the gel to avoid bubbles, attach it to the kit with 3M paper soaked with transfer buffer, and electrophoresis from the gel. The protein is transferred to the membrane.

By the way, when the thickness of the membrane used is thin, it is often difficult to put it correctly on the gel (gel), and there is a tendency that the membranes stick together and do not easily work. In this case, not only do you get a good signal, but the background tends to get messy. In addition, if the thickness of the membrane is excessively increased in order to improve the workability, the process cost increases, and there is a difficulty in controlling the membrane pore structure.

In particular, 3M paper (3M paper) is placed on the membrane and mounted on an electrophoresis kit for transfer. If the membrane and paper are integrated, the beginners can easily use it, and the transfer process can be shortened as well as excellent sensitivity. Under the assumption that a membrane having a high molecular weight is provided, the inventors have conducted a lot of research on this, and then use the electrospinning technique to integrate a membrane and a paper composed of nanofibers whose pore structure can be controlled and whose surface area is maximized for protein detection and diagnosis. The present invention was completed by discovering that the transfer membrane can be improved in sensitivity.

The purpose of the present invention is to improve the sensitivity by improving the workability and handling by integrating the surface of the nanofibrous layer with the surface area maximized by using the electrospinning technique of the membrane for Western blot used for protein detection and diagnosis, It is to provide a membrane that can provide user convenience.

In order to achieve this object, the present invention comprises the steps of dissolving a hydrophobic polymer in a solvent to prepare a spinning solution; Obtaining a nanofiber web of a hydrophobic polymer by spinning the spinning solution; Laminating the obtained nanofiber web to obtain a membrane for western blot; And laminating the membrane with a paper for western blot having an adhesive layer formed on one surface thereof to obtain an integrated membrane in which the membrane and the paper are integrally bonded to each other.

In addition, the present invention comprises the steps of dissolving a hydrophobic polymer in a solvent to prepare a spinning solution; Spinning the spinning solution on a surface of the Western blot paper on which an adhesive layer is formed by spinning the hydrophobic polymer nanofiber web; Laminating the obtained nanofiber web to provide a method of manufacturing an integral membrane for western blot comprising the step of obtaining an integral membrane in which the membrane and the paper are integrally bonded.

The present invention also provides a nanofiber / paper integrated membrane for western blot configured by applying or spraying an adhesive material on a paper, wherein the hydrophobic polymer nanofiber layer and the paper are adhesively bonded.

The present invention also provides a nanofiber / paper integrated membrane for western blot which prevents the separation of the nanofibers and the separation of the nanofiber layer and the paper by laminating the hydrophobic polymer nanofiber layer and the paper.

In addition, the present invention is prepared by laminating a nanofiber web obtained by electrospinning the spinning solution of a hydrophobic polymer material, the membrane having an average pore diameter of 0.1 to 0.5㎛, membrane thickness of 30 to 200㎛; And it provides a one-piece membrane for western blot, characterized in that it comprises a paper for Western blot bonded to one surface of the membrane via an adhesive layer.

Examples of the hydrophobic polymer include polyvinylidene fluoride (PVDF), nylon (nylon), nitrocellulose, polyurethane (PU), polycarbonate (PC), polystryene (PS), polyacrylonitrile (PAN), and polylatic (PLA). acid), PLGA, (polylactic-co-glycolic acid) PEI (polyethyleneimine), PPI (polypropyleneimine), PMMA (polymethylmethacrylate), PVC (polyvinylcholride), PVAc (polyvinylacetate), polystyrene divinylbenzene copolymer, etc. It may be configured by combining alone or in combination, there is no particular limitation on these.

The solvent is dimethyl formamide (di-methylformamide, DMF), dimethyl acetamide (di-methylacetamide, DMAc), THF (tetrahydrofuran), acetone (Acetone), alcohol (Alcohol), chloroform (Chloroform), DMSO (dimethylSO) sulfoxide), dichloromethane (dichloromethane), acetic acid (acetic acid), formic acid (formic acid), NMP (N-Methylpyrrolidone), characterized in that at least one member selected from the group consisting of water, and water.

The radiation method is any one selected from the group consisting of electrospinning, electrospray, electrobrown spinning, centrifugal electrospinning, flash-electrospinning, and the like. It is characterized by.

The laminating bond of the nanofiber layer composed of the hydrophobic polymer is preferably made by any one of a post-treatment process such as pressing, rendering, heat treatment, rolling, thermal bonding, ultrasonic bonding, and seam sealing tape.

Adhesives usable in the present invention are methanol, water, or products which are not decomposed during electrophoresis and are cured at room temperature or heat treatment, and are at least one member selected from silicon, epoxy, acrylic, urethane, and the like.

As said paper, if it is a paper for Western blots which is 3M company, and paper whose thickness is 100 micrometers or more, it is sufficient, and is not limited to a specific thing.

As described above, according to the present invention, it is possible to improve workability and handleability by integrating a nanofibrous layer and paper of which surface area is maximized by using an electrospinning technique for the Western blot membrane used for protein detection and diagnosis. There is an advantage that can provide convenience.

In addition, according to the present invention, the integrated membrane can be mass-produced at a low cost, and has an advantage that it can be utilized for protein separation, analysis, and various detections by providing excellent sensitivity compared to existing membranes.

The integrated membrane consisting of nanofibers and paper for Western blot according to the present invention first prepares a solution at a concentration capable of spinning a hydrophobic polymer, and transfers it to a spinneret, followed by electrospinning by applying a high voltage to a nozzle. In order to adhere to the paper, a curable adhesive is sprayed or coated to prepare a membrane for protein separation and detection, which is formed by laminating a nanofiber layer, paper, and the like.

Hereinafter, each step will be described in detail.

Preparation of Hydrophobic Polymer-containing Spinning Solution

Prepare a spinning solution by dissolving the hydrophobic polymer to a spinnable concentration using a suitable solvent. In the present invention, the hydrophobic polymer material is PVdF (polyvinylidene fluoride), Nylon, nitrocellulose, PU (polyurethane), PC (polycarbonate), PS (polystryene), PLA, PLGA, PEI (polyethyleneimine), PPI (polypropyleneimine), PMMA, PVC, PVAc, Polystylene divinylbenzene copolymer, etc. can be used alone or in combination. Prepare a spinning solution using a compatible solvent.

The amount of hydrophobic polymer in the spinning solution is preferably 5 to 50% by weight based on the total spinning solution, and less than 5% by weight is sprayed onto a bead rather than forming nanofibers to form a membrane. It is difficult, and when it exceeds 50 weight%, a viscosity may be high and it may be difficult to form a fiber because it is poor in spinning property. Therefore, the preparation of the spinning solution is not particularly limited, but it is preferable to control the morphology of the fiber to a concentration that is easy to form a fibrous structure.

Polymer Nanofiber Web Formation

The prepared spinning solution is transferred to a spin pack using a metering pump. At this time, a high voltage controller is used to apply a voltage to the spinning pack to perform electrospinning. At this time, the voltage used can be adjusted up to 0.5kV ~ 100kV, the current collector may be grounded or charged to the (-) pole, it is preferable to be composed of an electrically conductive metal, release paper and the like. In the case of the current collector plate, it is preferable to use a suction collector attached to smooth the focusing of the fiber during spinning.

In addition, the distance between the spin pack and the current collector is preferably adjusted to 5 to 50 cm. The amount of discharge during spinning is discharged at a discharge rate of 0.01 to 5 cc / hole.min per hole using a metering pump, and is preferably radiated in an environment with a relative humidity of 30-80% in a chamber that can control temperature and humidity during spinning.

Adhesive application step

The adhesive is sprayed onto 3M paper or drawing paper and uniformly applied so that no separation occurs between the nanofiber layer and the paper. The coating amount should be appropriately adjusted so that the adhesive does not come out of the fiber layer during thermocompression bonding, and the adhesive is preferably a thermosetting adhesive that is cured during heat treatment.

Laminating step of nanofibers and paper

The nanofibers made of the prepared hydrophobic polymer are laminated with various methods such as pressing, rolling, thermal bonding, ultrasonic bonding, and sim sealing tape by laminating with adhesive coated paper.

At this time, it is preferable to perform temperature at about 150 degreeC. That is, the adhesive must be cured by heat treatment so that it is not degraded by methanol or upon electrophoresis when performing protein analysis.

The thickness of the membrane is 30 to 200 µm and the average pore diameter is 0.1 to 0.5 µm. If the membrane thickness is less than 30 μm, Western bloat rigidity is low, and handling is poor. If the thickness is more than 200 μm, the manufacturing cost increases, which is not preferable. In addition, when the average pore diameter of the membrane is less than 0.1㎛ the process cost by post-treatment and the transfer time is delayed, if more than 0.5㎛ has a disadvantage that the accurate protein analysis can not be carried out low concentration of the transfer.

In particular, when laminating can be accompanied with a heat treatment, it is preferable to perform in a temperature range of 60 ~ 200 ℃ that the polymer is not melted. If less than 60 ℃ heat treatment temperature is low due to unstable fusion between nanofibers, it is difficult to perform a suitable western blot due to the separation of nanofibers during methanol pretreatment before performing the Western blot. In addition, when the heat treatment temperature exceeds 200 ° C., the pore structure is blocked by the melting of the hydrophobic polymer constituting the nanofibers, so that the protein is not transferred from an appropriate SDS-page, and thus, an accurate analysis does not occur.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, it will be apparent to those skilled in the art that the embodiments of the present invention are merely for illustrating the present invention, and the scope of the present invention is not interpreted to be limited by these examples.

(Example 1)

1. Preparation of PVdF Nanofiber Layer

Hydrophobic polymer PVdF (Kyner 761) was dissolved in the solvent DMAc to 20% by weight. The prepared solution was transferred to a spinning nozzle using a metering pump, and electrospinning was performed at room temperature and atmospheric pressure at an applied voltage of 25 kV, a distance of 20 cm between the spinneret and the current collector, and a discharge amount of 0.05 cc / g per minute. A scanning electron micrograph of the PVdF nanofiber layer was shown in FIG. 1.

As shown in FIG. 1, the fiber diameter constituting the obtained PVdF nanofiber layer was mostly less than 1 μm, indicating an average of about 400 nm. At this time, the thickness of the membrane composed of spun nanofibers was about 80㎛. The membrane was subjected to lamination using a roller heated at 140 ° C. to a thickness of about 60 μm, wherein the average pore diameter (diameter) was 0.45 μm.

2. Preparation of Integral Membrane

A thermosetting silicone adhesive was sprayed onto a paper for Western blot provided by 3M, to prepare a paper coated with an adhesive layer. The PVdF nanofiber layer prepared by the method of 1 was laminated on the thus prepared paper. The laminated composite membrane was pressed with a laminating machine composed of a roller at 145 ° C. to obtain an integrated membrane in which paper and nanofiber layers were integrally combined.

Figure 2 is a scanning electron micrograph showing the cross section of the integral membrane obtained according to the present embodiment, as shown in Figure 2, it can be seen that the nanofiber layer constituting the membrane is firmly bonded by fusion with 3M paper.

3. Western blot test

Western blot was performed using the integral membrane sample prepared in 2 above. First, the prepared samples were cut into pieces of 6 × 8 cm in width and length, and then immersed in 100% methanol for 5 minutes to enable hydrophobic interaction with proteins in the gel.

The activated membrane was transferred to a 1 ㅧ transfer buffer and left for 10 minutes. At this time, the configuration of the 1 ′ transfer buffer (1 ′ transfer buffer) was 3.03 g / L trisma-base, 14.4 g / L glycine, 20% methanol (200 ml / L). The gel to be transferred is slightly wetted with 1 ㅧ transfer buffer and carefully placed on the membrane to prevent bubbles from forming.

The transfer was performed for 1 hour at 100V using a Mini-gel transfer kit, and the transfer tank was placed in ice to prevent heat generated at this time. After the transfer is complete, dismantle the device, remove the membrane and gently squeeze to 1xTBST. TBST consisted of 0.2M Tris pH 8 (24.2g Trisma base), 1.37M NaCl (80g NaCl), and Adjust pH7.6 by conc HCl.

At this time, the concentration of purified protein antigen was 20, 10, 5, 2.5, 1 ㎍, 10% SDS-page gel was used. The total transfer time was about 1 hour 40 minutes and the blocking time was 1 hour 30 minutes. The first antibody was β-actin (Santa cruz, sc-47778). Ab dilution: 1: 5000 was performed at -4 ℃ for about a day. This transfer was confirmed the protein expression using the X-ray film, the results are shown in Figure 5 compared to the conventional non-integral case.

As shown in Figure 5, the case of the present invention can be seen that the size of the blot more clearly than the conventional. This is interpreted because the case of the present invention works more sensitively than in the prior art.

More specifically, in the conventional case, the membrane is placed on the gel (gel) and the paper is placed on it again, and then mounted on an electrophoresis kit to perform electrophoresis for about 1 hour, so that the membrane and If the adhesion to the gel is weak or there is space in the kit when the gel is in contact with the membrane, a flow between the paper and the membrane may occur, making it difficult to obtain an accurate protein transfer. In addition, even when the same membrane is used, the paper and the membrane are integrated with each other, so that the protein transfer paper fixes the membrane, thereby enabling the transfer of the protein to a more accurate position. For this reason, it is expected that more accurate and improved sensitivity is obtained compared to the prior art.

In the case of Figure 6, as shown in Figure 5 above, if there is a space in the kit or the gel and the membrane did not contact correctly, in the prior art there is a possibility that the membrane is caused to flow or move much more than the one-piece background.

In addition, by integrating the membrane and the paper as shown in Figure 6, even a beginner can easily perform Western blotting without a subsequent process, and when the PVdF membrane activated by methanol on the gel as in the conventional method, the membrane is bent due to no power It is possible to fix the gel and the membrane in the correct position through several trials and errors due to difficulty in forming, foaming, or matching exactly.However, the membrane is simply and easily fixed by integrating the membrane and paper and activating with methanol. The hard paper is raised to prevent bubbles from being generated and the position can be fixed with a single operation, resulting in a low background and high signal. Thus, the integrated type could provide higher sensitivity than the prior art.

(Example 2)

The MMP-2 protein was analyzed in the same manner as in Example 3 using the monolithic membranes prepared by Examples 1 and 2, and the results are compared with those of the conventional non-integral case. Indicated. Actin used at this time was carried out to see if the amount of protein walking the same.

As shown in Figure 6, in the case of the present invention it can be seen that the analysis results appear more clearly than the background compared to the conventional, in the case of Figure 6, as in the case of FIG. In case of inaccurate contact, it is considered that the conventional separation type tends to cause the membrane to flow down or move, causing much more background.

According to the present invention, by integrating the membrane and paper, even beginners can easily perform Western blotting without a subsequent process.

In other words, when the PVdF membrane activated by methanol is placed on the gel as in the conventional method, it is difficult to bend, bubble, or match exactly due to the force of the membrane, and only fix the gel and the membrane in the correct position only through repeated trials and errors. In the present invention, the membrane and the paper can be integrated and activated to easily and easily fix the membrane on the gel, and since the hard paper is raised, no bubbles are generated, and the position and the like can be simply fixed by a single operation. You can get low background and high signal. Therefore, there is an advantage that the integrated type can provide a higher sensitivity than the prior art.

1 is a scanning electron micrograph showing a PVdF nanofiber web obtained according to the present invention.

Figure 2 is a scanning electron micrograph showing a cross section of the integral membrane prepared by one embodiment of the present invention.

3 is an analysis result of Western blot using the integrated membrane prepared according to an embodiment of the present invention.

4 is an analysis result of Western blot using the integrated membrane prepared by another embodiment of the present invention.

Claims (11)

Dissolving the hydrophobic polymer in a solvent to prepare a spinning solution; Obtaining a nanofiber web of a hydrophobic polymer by spinning the spinning solution; Laminating the obtained nanofiber web to obtain a membrane for western blot; And laminating the membrane with a paper for Western blot having an adhesive layer formed on one surface thereof to obtain an integrated membrane in which the membrane and the paper are integrally bonded. Dissolving the hydrophobic polymer in a solvent to prepare a spinning solution; Spinning the spinning solution on a surface of the Western blot paper on which an adhesive layer is formed by spinning the hydrophobic polymer nanofiber web; Laminating the obtained nanofiber web to obtain an integral membrane in which the membrane and the paper are integrally bonded. The method according to claim 1 or 2, The hydrophobic polymer material is polyvinylidene fluoride (PVDF), nylon (nylon), nitrocellulose (nitrocellulose), PU (polyurethane), PC (polycarbonate), PS (polystryene), PLA (polylatic acid), PLA (polyacrylonitrile), PLGA, (polylactic-co-glycolic acid) in the group consisting of polyethyleneimine (PEI), polypropyleneimine (PPI), polymethylmethacrylate (PMMA), polyvinylcholride (PVC), polyvinylacetate (PVAc), and polystylene divinylbenzene copolymer A method for producing an integrated membrane for western blot, characterized in that it is composed by combining any one or two or more selected. The method according to claim 1 or 2, The solvent is dimethyl formamide (di-methylformamide, DMF), dimethyl acetamide (di-methylacetamide, DMAc), THF (tetrahydrofuran), acetone (Acetone), alcohol (Alcohol), chloroform (Chloroform), DMSO (dimethylSO) Integral membrane for western blot, characterized in that at least one member selected from the group consisting of sulfoxide, dichloromethane, acetic acid, formic acid, N-Methylpyrrolidone (NMP), fluorinated alcohols, and water Manufacturing method. The method according to claim 1 or 2, The diameter of the nanofibers forming the nanofiber web is 50 to 1000nm, characterized in that the manufacturing method of the integral membrane for western blot. The method according to claim 1 or 2, The radiation method is any one selected from the group consisting of electrospinning, electrospray, electroblown spinning, centrifugal electrospinning, and flash-electrospinning. Method for producing an integrated membrane for western blot, characterized in that. The method according to claim 1 or 2, Laminating in each step is a method of manufacturing a unitary membrane for western blot, characterized in that carried out by any one method selected from pressurization, rendering, rolling, thermal bonding, ultrasonic bonding, seam sealing tape method. The method according to claim 1 or 2, The content of the hydrophobic polymer material with respect to the whole spinning solution is 5 to 50% by weight of the manufacturing method of the integral membrane for western blot. The method according to claim 1 or 2, The laminating process in each step is a method of manufacturing an integrated membrane for western blot, characterized in that performed with heat treatment in the temperature range of 60 ~ 200 ℃. A membrane formed of a nanofiber web of hydrophobic polymer material; And An integrated membrane for western blot consisting of paper for western blot adhered to one surface of the membrane via an adhesive layer. A hydrophobic polymer membrane prepared by laminating a nanofiber web obtained by electrospinning a spinning solution of a hydrophobic polymer material and having an average pore diameter of 0.1 to 0.5 µm and a thickness of 30 to 200 µm; And An integrated membrane for western blot, characterized in that it comprises a paper for western blot to be bonded and integrated through an adhesive layer on one surface of the membrane.

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