KR20110035454A - Nano-fibered membrane for western blot and manufacturing method of the same - Google Patents

Abstract

PURPOSE: A nano-fiber membrane for western blot and a method for manufacturing the same are provided to easily implement a protein separation process, a protein analyzing process, and various detection processes using a spinning method. CONSTITUTION: A hydrophobic polymer material is dissolved in a solvent in order to obtain a spinning solution. The spinning solution is used for a spinning method in order to obtain a hydrophobic polymer nano-fiber web. The hydrophobic polymer material nano-fiber web is laminated to obtain a nano-fiber membrane for western blot. The hydrophobic polymer material is a composite based on one, two, or more selected from a group including polyvinylidene fluoride, nylon, nitrocellulose, polyurethane, polycarbonate, polystyrene, polylactic acid, polyacrylonitrile, polylactic-co-glycolic acid, polyethyleneimine, polypropyleneimine, polymethylmethacrylate, polyvinylchloride, polyvinylacetate, and polystyrene divinylbenzene copolymer.

Description

Nano-fibered Membrane for Western Blot and Manufacturing Method of the Same}

The present invention relates to a membrane for a western blot composed of nanofibers and a method for manufacturing the same, and specifically, a Western blot composed of an average fiber diameter of 50 to 1000 nm and an average pore diameter of 0.1 to 0.5 μm by electrospinning. It relates to a transfer membrane for a lot and a method of manufacturing the same.

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. This is the most effective way to find out.

Currently, to detect a specific substance, chromatography such as gas chromatography (GC) or high performance liquid chromatograph (HPLC), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (Immunohis technology) and Methods such as DNA analysis have been used, and in the case of proteins, a method of isolating and analyzing specific proteins by antigen-antibody reaction using Western blot is generally known.

Western blot mixed the protein extracted from the cells or tissues with the sample buffer and placed it in a molecular sieve made of acrylamide, followed by electrophoresis, and the SDS (sodium dodecylsulfate, SDS) contained in the sample buffer. A substance called -page) charges the entirety of the protein and attracts the protein towards the positive. At this time, molecular sieves interfere with the progress of the protein, and small molecules move quickly and large molecules move slowly to form bands of various sizes. At this time, the membrane is placed on a gel separated according to the size and electricity is supplied. Upon shedding, the protein is transferred to the membrane in a separated state. Herein, a method of binding an antibody to a specific protein to be detected and binding a second antibody specific for the antibody to X-ray image of the reaction caused by color development or fluorescence. Figure 1 generally shows the process of protein detection using Western blot method.

The transfer membrane used here uses nitrocellulose, nylon, polyvinylidene fluoride (PVdF), etc., which are easy to hydrophobic interaction with proteins, and has an average pore diameter of 0.2 ㎛ to 0.45. It consists of micrometers. 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. 2 shows a method of forming a film structure prepared by a typical phase transfer method.

Therefore, the inventors of the present invention have a low cost, simple manufacturing method using the spin technique, artificial control of the pore structure, and developed a membrane composed of nanofibers with a maximum surface area, thereby increasing sensitivity for protein detection and diagnosis. The present invention has been completed by making a good transfer membrane.

SUMMARY OF THE INVENTION An object of the present invention is to provide a highly sensitive membrane composed of nanofiber webs with a surface area maximized by using a spinning method for Western blot membranes used for protein detection and diagnosis, and a method of manufacturing the membrane.

In order to achieve this object, according to the present invention, a method for producing a membrane for Western blot, comprising the steps of dissolving a hydrophobic polymer in a solvent to prepare a spinning solution; Obtaining a hydrophobic polymer nanofiber spinning web by spinning the spinning solution; Laminating the obtained nanofiber spinning web (laminating) is provided a method for producing a membrane for Western blot comprising the step of obtaining a membrane for Western blot.

According to the present invention, there is also provided a membrane for a Western blot prepared according to the above method, wherein the membrane for a Western blot is characterized by having an average pore diameter of 0.1 to 0.5 µm, a thickness of 30 to 200 µm, and a porosity of 60% or more. do.

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, pressing, rendering, heat treatment, rolling, thermal bonding, ultrasonic bonding, and seam sealing tape.

Western blot membrane prepared by the present invention can be mass-produced at low cost because the manufacturing process is simple, and can be used for protein separation, analysis and various detection by providing excellent sensitivity compared to the existing membrane.

For example, a membrane composed of a nanofiber web for western blot according to the present invention first dissolves a hydrophobic polymer in an appropriate solvent to prepare a solution at an irradiable concentration, transfers it to a spinneret, and then applies a high voltage to the nozzle to perform electrospinning ( electrospinning) and laminating the same to prepare a membrane for protein separation and detection consisting of an average pore diameter of 0.1 to 0.5 µm, an average fiber diameter of 50 to 1000 nm, and a membrane thickness of 30 to 200 µm.

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 the hydrophobic polymer material in the spinning solution is about 5 to 50% by weight is appropriate, less than 5% by weight is difficult to form a membrane by spraying onto the beads (bead) rather than forming nanofibers, 50% by weight In the case of more than%, a viscosity may be high and it may be difficult to form a fiber because of poor 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 by discharging at 0.01 ~ 5cc / hole.min per hole using a metering pump, and spinning in an environment with a relative humidity of 30-80% in a chamber that can control temperature and humidity during spinning. desirable.

Polymer Nanofiber Web Laminating Step

The nanofiber web composed of the prepared hydrophobic polymer is laminated by various methods such as crimping, rolling, thermal bonding, ultrasonic bonding, and sim sealing tape, and has a membrane thickness of 30 to 200 µm and an average pore diameter of 0.1 to 0.5 µm. To be

If the thickness of the membrane is less than 30㎛ western blotting stiffness is poor handling, if the thickness exceeds 200㎛ 30 ~ 200㎛ is suitable because the manufacturing cost increases. In addition, the average membrane pore size is 0.1 ~ 0.5㎛, if less than 0.1㎛ post-treatment process cost increases, the transfer time is delayed, if more than 0.5㎛ transporter concentration is low, accurate protein analysis can not be achieved There is this.

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 the appropriate western blot due to the separation between nanofibers during methanol pretreatment prior to 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. These examples are only intended to illustrate the invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

(Example 1)

PVdF (HSV 900) consisting of a hydrophobic polymer homo was dissolved in solvent DMAc to 20% by weight. The prepared solution was transferred to a spinning nozzle using a metering pump, and spinning PVdF nanofibers at room temperature and atmospheric pressure with an applied voltage of 25kV, a distance of 20cm between the spinneret and the current collector, and a discharge amount of 0.05cc / g · holl per minute. Got the web.

The thickness of the nanofiber spinning web obtained by spinning was about 120 μm, and the PVdF spinning web was laminated using a roller heated to 145 ° C. to manufacture a PVdF membrane. The thickness of the membrane after lamination was about 80 μm. The average pore size showed 0.45 µm.

Figure 3 shows a scanning electron micrograph of the PVdF membrane obtained in Example 1. As shown in FIG. 3, the fiber diameter of PVdF nanofibers was mostly less than 1 μm, and the average was about 500 nm. It can be seen that the pore diameter is relatively uniform and the structure is formed in a three-dimensional open pore channel.

(Examples 2 and 3)

Examples 2 and 3 were prepared by mixing PVdF 761 alone (Example 2) composed of a hydrophobic polymer homopolymer (Example 2) and a copolymer of PVdF 2801 with PVdF 761 at 50: 50% by weight (Example 3). Electrospinning was carried out in the same manner as the method.

The prepared samples of Examples 2 and 3 were subjected to TGA, XRD, SEM, DSC analysis for comparison with the sample of the comparative example. Thermal analysis showed almost the same results for the samples prepared by Comparative Example and Example, showing typical results of PVdF polymer.

(Comparative Example)

For comparison, Western blot was performed in the same manner as in Example 3 using a PVdF membrane of Pall (PALL CO., LTD., BioTrace ™ PVDF), a commercially available membrane prepared by using a phase separation method.

Membrane Structure Analysis

Structural analysis of the PVdF membranes prepared in Examples 1 to 3 and the commercialized membranes prepared in Comparative Examples were performed through DSC, XRD, TGA, and SEM. As a result of the SEM analysis, in the case of the comparative example, as shown in Fig. 4 showing a scanning electron micrograph of the prepared membrane, the pore diameter was not uniform, and the pore structure also showed a closed pore structure. .

Referring to FIG. 5 showing the result of thermogravimetric analysis (TGA), both the comparative example and the embodiment of the commercialized membrane showed the same behavior that does not decompose in the air up to about 500 ℃, the examples of the present invention It can be seen that it is somewhat thermally stable compared with that.

On the other hand, the porosity (porosity) of each membrane prepared by Example 1 and Comparative Example of the present invention was measured by the following equation (1) by cutting each 1cm in width.

..........(One)

는 PVdF의 밀도로 1.76g/㎤ 이며,

는 계산된 값이다. here,

Is the density of PVdF of 1.76 g / cm 3,

Is the calculated value.

The porosity obtained by the above formula is 73.3% in Example 1 and about 67% in Comparative Example, indicating that the workability of the membrane prepared by the method of Example 1 of the present invention is about 10% larger. Confirmed.

Western Blot Test

Western blot was performed using the sample of Example 1 and Comparative Example.

First, the samples prepared in Example 1 and Comparative Example were precut into 8 cm ㅧ 9 cm in width and length, respectively, and then subjected to about 100% methanol so that hydrophobic interaction between the protein in the gel and the membrane was possible. It was activated by soaking for 5 minutes.

This activated membrane was transferred to transfer buffer (1 ㅧ transfer buffer) and left for 10 minutes. The transfer buffer was composed of 3.03 g / L trisma-base, 14.4 g / L Glycine, and 20% methanol (200 ml / L). Lightly dampen the gel to be transferred with transfer buffer and place it on the membrane carefully to avoid bubbles. After the gel and the membrane are in close contact with each other, the 3M paper previously wetted with transfer buffer solution is mounted on the transfer kit.

Transfer was carried out for 1 hour at 100V using a Mini-gel transfer kit, the transfer tank was carried out in ice to block the heat generated at this time. After transfer, disassemble the device, remove the membrane and gently rinse with 1xTBST (Tris-buffered saline with 0.05% tween 20). TBST consists of 0.2M Tris pH 8 (24.2g Trisma base), 1.37M NaCl (80g NaCl), and Adjust pH 7.6 by conc HCl.

The total protein concentrations of oral epithelial carcinoma KB cell line were 20, 10, 5, 2.5, 1㎍, and 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 protein to be detected was β-actin, and the first antibody was β-actin antibody (Santa cruz, sc-47778) obtained from the mouse, and diluted 1: 5000 to react with the transfer membrane at 4 ° C. for about 1 day. I was. After that, it was reacted with goat anti-mouse IgG-HRP (Santa cruz, sc-2005, an antibody prepared by injecting mouse immunoglobin into goats), which is a secondary antibody (Secondary Antibody) to which horseradish peroxidase is bound, and then horseradish peroxiase ( Luminol is fluoresced by oxidizing the hydrogen peroxide solution and Luminol Enhancer Solution (hydrogen peroxide decomposed by hydrogen peroxidase), which are substrates for horseradish hydrogen peroxidase; LF-QC1010, ABFRONTIER, Korea) was added and reacted for 1 minute. The transfer membrane reacted with the substrate was exposed to the X-ray film for 2 minutes to confirm the expression of β-actin protein.

Figure 6 shows the results of Western blot using the membrane prepared by Example 1 and Comparative Example.

As shown in Figure 6, the case of Example 1 can be seen that the reaction band appears more clearly and clearly than the comparative example. This is because the membrane of the embodiment of the present invention has a more uniform pore diameter than the comparative example and has a larger specific surface area due to high porosity, thereby showing excellent sensitivity.

In the above, the present invention has been illustrated and described with reference to specific preferred embodiments, but the present invention is not limited to the above-described embodiments and the general knowledge in the art to which the present invention pertains is not limited to the spirit of the present invention. Various changes and modifications will be made by those who possess.

1 is a flow chart showing a method for detecting a specific protein by Western blot method using a PVdF membrane,

Figure 2 is a flow chart showing a method of manufacturing a membrane by a conventional general phase transition method.

3 is a scanning electron micrograph of the PVdF membrane prepared by Example 1 of the present invention; (a) 1k, (b) 10k x magnification,

4 is a scanning electron micrograph of the membrane prepared by the comparative example; (a) 3k, (b) 10k x magnification,

5 shows the results of TGA analysis in air of membranes prepared by Examples and Comparative Examples of the present invention; (a) Comparative Example, (b) Example 1 (PVdF HSV 900), (c) Example 2 (PVdF 761), (d) Example 3 (PVdF 761/2801),

Figure 6 shows the results of Western blot evaluation of each membrane prepared by Example 1 and Comparative Example of the present invention.

Claims (9)

In the manufacturing method of the membrane for Western blot, Dissolving the hydrophobic polymer in a solvent to prepare a spinning solution; Obtaining a hydrophobic polymer nanofiber web by spinning the spinning solution; And laminating the obtained nanofiber web to obtain a membrane for western blot. The method of claim 1, 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 Method for producing a membrane for Western blot, characterized in that it is composed by combining any one or two or more selected. The method of claim 1, The solvent is dimethyl formamide (di-methylformamide, DMF), dimethyl acetamide (di-methylacetamide, DMAc), THF (tetrahydrofuran), acetone (Acetone), alcohol (Alcohol), chloroform (Chloroform), DMSO (dimethylSO) 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), fluorine-based alcohols, and water Manufacturing method. The method of claim 1, The diameter of the nanofibers forming the nanofiber web is a method for producing a membrane for Western blot, characterized in that 50 to 1000nm. The method of claim 1, The radiation method is any one selected from the group consisting of electrospinning, electrospray, electroblown spinning, centrifugal electrospinning, and flash-electrospinning. Method for producing a membrane for Western blot, characterized in that. The method of claim 1, The laminating is a method for producing a membrane for Western blot, characterized in that performed by any one method selected from the crimping, pressurization, rendering, rolling, thermal bonding, ultrasonic bonding, seam sealing tape method. The method of claim 1, The amount of the hydrophobic polymer material to the total spinning solution is a manufacturing method of the membrane for Western blot, characterized in that 5 to 50% by weight. The method of claim 1, The laminating process is a manufacturing method of the membrane for Western blot, characterized in that performed with heat treatment in the temperature range of 60 ~ 200 ℃. The western blot membrane prepared according to any one of claims 1 to 8, wherein the western blot membrane has an average pore diameter of 0.1 to 0.5 µm, a membrane thickness of 30 to 200 µm, and a porosity of 60% or more. Membrane for Lot.

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