JP2009156767A - Protein array substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein array capable of accurately detecting and analyzing the interactions among proteins or the interaction among proteins and compounds, other than proteins, and providing a protein array substrate capable of detecting interactions with proteins, having moreover, large molecular weights such as antibodies in addition. <P>SOLUTION: A frame body is mounted on a substrate, and a monomer solution is made to flow in and gelate under a water-vapor atmosphere and then dried after gelation, and washing under a water-vapor atmosphere to prepare the protein array substrate. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a protein array substrate used for detecting and analyzing an interaction between a protein and a protein, or an interaction between a protein and a biomolecule or a compound other than the protein by an optical means.

Protein arrays that use water-containing gels that do not cause protein denaturation or deactivation in a relatively close state to the body in order to study interactions between proteins, or interactions between proteins and low-molecular-weight compounds and other biomolecules. It has been known.
On the other hand, a protein array of such a hydrogel type that detects biomolecules that bind to the immobilized protein based on the intensity of transmitted light is also known. There is no need to label the molecule, and it is possible to quantitatively detect the interaction between the non-labeled biomolecule and the immobilized protein.

  A method for producing such a hydrogel type protein array substrate is that a gel monomer mixed solution is dropped on a substrate, and a cover member such as a cover glass used for microscopic observation is placed thereon to polymerize and gel, The array substrate was prepared by removing the flat plate. After the gelation, the substrate was washed with water to remove unpolymerized monomers, and then dried (Patent Application No. 2006-546655, FIG. 1). However, such a conventional method for producing a protein array substrate has the following problems.

  That is, in the conventional method, a monomer solution is dropped onto a substrate and a cover member is placed thereon to gel, and gel polymerization is performed while the monomer solution at the end is in contact with the outside air. As the solution leaks out of the cover member, the end of the gel is raised, or unevenness occurs due to the degree of gelation and the degree of drying depending on the location. It was difficult to produce a gel substrate having a high thickness and high flatness. For this reason, when using such an array in which proteins are immobilized on a substrate to detect and analyze interactions with other proteins or other substances by optical means, it is caused by non-uniformity of the gel substrate. As a result, variations in signal intensity occurred, making it difficult to interpret and analyze the measurement results. In addition, according to the above conventional method, it is difficult to form a thick gel, so that the amount of protein that can be immobilized per unit area of the substrate is small, and the number of molecules that interact with it is also small. As a result, there was a problem that the weak interaction could not be detected.

  Furthermore, in the case of the conventional method, if the concentration of the gel monomer is lowered or the content of the cross-linking agent is changed, the polymerization of the monomer becomes difficult, and a part of the gel is dried, which makes it difficult to form the gel. The yield and quality reproducibility are poor, and it is difficult to produce a gel substrate having a gel with a large pore size. For this reason, it is difficult for a protein having a large molecular weight such as an antibody to diffuse into the gel, and as a result, it is difficult to detect an interaction with such a large protein. In addition, as a result of photographing a spot on which a protein was immobilized on a gel substrate prepared by a conventional method with ultraviolet transmitted light, it was observed that the spot was uneven at the periphery and inside. Is not fixed uniformly, and it has become clear that there is a problem in analyzing the measurement results.

  An object of the present invention is to eliminate the problems of the conventional methods described above in a protein array of a type that measures transmitted light without labeling a substance to be measured. Providing a protein array that can accurately detect and analyze the interaction of proteins with biomolecules and compounds other than proteins, and to provide a protein array substrate that can detect interactions with proteins such as antibodies Is.

As a result of earnest research to solve the above problems, the present inventor, in particular, uses a frame as a protein array substrate preparation member, performs gelation in a water vapor atmosphere, and dries after washing with gelled water. It was found that the above-mentioned problems can be solved by carrying out in a steam atmosphere, and the present invention has been completed.
That is, the present invention is as follows.

(1) A substrate, a frame body placed on the substrate and containing a gel monomer-containing solution, and a cover member that covers the upper portion of the frame body. On the base plate, the frame body and the cover member An apparatus for producing a protein array substrate, wherein a space for polymerizing and gelling a gel monomer is formed in a state of being blocked from outside air.
(2) The apparatus for preparing a protein array substrate according to (1) above, wherein the frame is formed of a material having adhesion to the substrate.
(3) The apparatus for producing a protein array substrate according to (2) above, wherein the frame is made of silicon rubber or silicon film.
(4) The apparatus for preparing a protein array substrate according to any one of (1) to (3) above, wherein the cover member is a water-repellent flat plate.
(5) The apparatus for producing a protein array substrate according to (5) above, wherein the gel monomer-containing solution contains polyacrylamide, a crosslinking agent thereof, an amino group-containing polymer, and a polymerization accelerator.
(6) A frame body is placed on a substrate, a gel monomer-containing solution is allowed to flow into the frame body, and then the gel monomer is polymerized and gelled in a state of covering the frame body with a cover member and blocking from the outside air. A method for producing a protein array substrate.
(7) The method for producing a protein array substrate according to (6) above, wherein the gel monomer-containing solution contains polyacrylamide, a crosslinking agent thereof, an amino group-containing polymer, and a polymerization accelerator.
(8) The method for producing a protein array substrate according to (6) or (7) above, wherein the polymerization of the gel monomer is performed under a constant temperature condition in a water vapor atmosphere.
(9) Preparation of the protein array substrate according to (8) above, wherein the gel monomer is polymerized and then washed with water, and further, moisture on the surface of the gel substrate is removed in a container under a water vapor atmosphere under a constant temperature condition. Method.
(10) A protein array, wherein a protein is immobilized on a protein array substrate produced by the production method according to any one of (6) to (9) above.

  In the present invention, a frame is used as an apparatus for producing a protein array substrate. The frame is placed on the substrate, and the gel monomer mixed solution is allowed to flow into the frame, and the cover is placed on the gel. By doing so, it is possible to prevent the gel monomer solution from leaking out of the cover member as in the conventional method, and the end portion is raised, and the degree of gelation and the degree of drying differ depending on the location. In addition, it is possible to eliminate unevenness of the gel surface by polymerizing the gel monomer under a constant temperature condition in a container in a water vapor atmosphere. As a result, the flatness of 0.1 mm to 0.5 mm in thickness is obtained. A high gel substrate can be produced. Since such a thick gel substrate can be produced, the amount of protein that can be immobilized increases, and as a result, even a biomolecule with a weak interaction and a small number of adsorption can be detected.

  In addition, by using the frame, it is possible to make the gel monomer polymerization process constant by preventing the contact between the gel monomer mixed solution and the outside air before solidification and performing gelation in the container, As a result, gels with various concentrations and cross-linking agent contents can be formed. On the other hand, if a cover member treated with water repellency is used in addition to this, it is possible to prevent the gel from collapsing, which was easy to occur when removing the cover member after polymerization. A gel substrate having a large pore size can be produced. As a result, even a biomolecule having a large molecular weight such as an antibody can be easily diffused into the gel, and the interaction with the immobilized protein can be detected. Moreover, as a result of making it possible to make the conditions of the gel monomer polymerization process constant, the yield is improved, and the reproducibility of quality including flatness is improved. Furthermore, in the present invention, since gel formation and drying after washing in water are performed in a water vapor atmosphere, the gel interior is not substantially dried, and a uniform moisture state is maintained. It is possible to produce a uniform protein spot with little unevenness at the periphery and inside.

By the above improvement, in the present invention, in the hydrogel type protein array for measuring the interaction between proteins, or the interaction between proteins and biomolecules such as bioactive substances, and the like, particularly by transmitted light, It is possible to provide a hydrogel-type protein array that can accurately detect and analyze the above interaction even when it is weak, and that can detect interactions with a wide range of protein and biomolecules.
In other words, most current protein arrays need to label biomolecules for detection, and have problems with reproducibility and quantification, and use transmitted light that does not require such labeling. The protein array used for the measurement method of the type also has a problem based on the non-uniformity of the gel surface, etc., but according to the present invention, the interaction between biomolecules can be accurately and quantitatively detected without labeling. Possible protein arrays can be realized.

The apparatus for producing a protein array substrate of the present invention comprises a substrate, a frame body placed on the substrate, and a cover member that covers the frame body. The material of the transparent substrate is not particularly limited, and examples thereof include glass, quartz glass, plastic, and the like, but those made of a material having no or low ultraviolet ray absorbing ability are preferable.
The frame is made of a □ -shaped member opened up and down, and is placed on a transparent substrate to store a gel monomer solution and form a reaction tank for polymerizing the gel monomer (FIG. 2). The material of the frame is not particularly limited as long as it is a material that does not cause liquid leakage when placed on a transparent substrate. However, a water-repellent material is preferable, and examples thereof include silicon rubber. The cover member is a flat plate member that covers the frame, and has a function of sealing the formed reaction tank. Examples of the material include glass and plastic, but the surface is water-repellent. It is preferable. The water repellent treatment is performed, for example, by coating the surface with a water repellent silane.

  Although the dimensions of these members can be freely changed, in the case of a substrate, for example, the length is 25 to 40 mm and the width is 40 to 80 mm. The vertical and horizontal dimensions of the frame are smaller than those of the substrate, but are not particularly limited as long as the storage layer can be formed by placing, but the thickness in the vertical direction is set to 0.1 to 0.5 mm, for example. Further, the width of each side of the frame body is not particularly limited as long as it is a dimension that causes liquid leakage and that can stably place the cover member and thereby seal the inside, but is set to, for example, 5 mm to 10 mm. Is preferred. The cover member should just be a dimension which can be mounted in a frame and can form sealed space.

Hereinafter, the process of producing a protein array substrate using these members will be described in detail.
(1) First, a frame is placed on a transparent substrate, and the gel monomer solution is allowed to flow into the formed storage tank. Next, a cover member is placed on the frame (FIG. 2). At this time, it is preferable not to form a space between the lower surface of the cover member and the upper surface of the gel monomer solution.
As a result, the monomer solution is prevented from contacting the outside air during storage and polymerization, and therefore, as in the conventional method, the end side of the monomer solution is not covered by the cover member, and thus is in contact with the outside air. It is possible to prevent a decrease in the flatness of the gel surface caused by non-uniformity in the degree of progress of the polymerization reaction.
The gel monomer solution used includes a component that forms a hydrogel, and includes a crosslinking agent, a reaction accelerator, and the like in addition to the hydrogel-forming monomer.

  Examples of the water-containing gel include a crosslinked polyacrylamide gel. When the water-containing gel is a crosslinked polyacrylamide gel, the monomer solution contains acrylamide as a monomer and N, N′-diallyl as a crosslinking agent. L-tartaric acid diamide or bisacrylamide, their polymerization accelerators TEMED and ammonium persulfate, and amino group-containing polymers such as poly-L-lysine are included, but other polymer molecules may be included. This amino group-containing polymer is used to immobilize the protein by polymerizing the gel and then forming an amide bond between the C-terminus of the protein and the amino group of the polymer.

  In the present invention, gel formation is possible even when a solution having a low monomer concentration is used. For example, when the crosslinked polyacrylamide gel is formed, the concentration of acrylamide in the solution may be 5% by weight. Gel formation is possible. Therefore, the concentration of acrylamide is adjusted to 5-10%.

(2) Next, the monomer solution was accommodated in the frame on the transparent substrate, and the gel-forming substrate covered with the cover member was placed in a water vapor atmosphere container, that is, a Kim wipe sufficiently containing pure water. The whole container is placed in a constant temperature incubator or the like and placed at a constant temperature, that is, at a constant reaction temperature, to carry out a monomer polymerization reaction (FIG. 3). The reaction temperature may be a temperature suitable for the polymerization of the monomer. However, when a polyacrylamide gel is formed, the reaction temperature is 20 to 30 ° C.
In this process, the polymerization reaction is sealed and performed in a water vapor atmosphere, so that it is completely blocked from the outside air and performed at a constant temperature, so that the conditions of the polymerization process remain constant at the optimum conditions for gel formation. it can. For this reason, a gel yield improves and the reproducibility of quality including the flatness of a gel improves.

(3) After the gel is formed by the polymerization reaction, the cover member and the frame are sequentially removed, and the transparent substrate on which the gel is formed is washed with water to remove residual components such as unreacted monomers.
In this case, the cover member is treated with water repellent. Alternatively, by further forming the frame body from a water-repellent material, it is possible to effectively prevent the gel from collapsing during removal, which frequently occurred in the conventional method.

(4) Next, in order to make it easy to absorb the protein to be immobilized in the gel, moisture is removed from the surface of the gel substrate that has been washed with water, thereby producing a substrate for a protein array.
In the present invention, surface moisture is removed by placing the water-washed gel substrate in a container that has been previously filled with a Kimwipe that contains sufficient pure water, and sealing it to achieve a water vapor atmosphere. Was carried out by allowing it to stand for about 16 hours in a constant temperature incubator set at 25 ° C. (FIG. 4). As a result, moisture is retained inside the gel, and the dry state of the surface can be made uniform. As a result, unlike the conventional method, drying unevenness of the gel surface does not occur, or drying does not proceed partially to the inside of the gel, and therefore, variation due to the location of the protein adsorption amount to be immobilized is prevented, The reliability of data obtained when used as a protein array is improved.

(5) Proteins are sequentially spotted on the protein array substrate prepared above to produce a protein array. In the present invention, the spotted protein is quickly absorbed into the gel, formed by the three-dimensional stitch structure constituting the gel, trapped in pores filled with an aqueous medium, and then with the amino group. Immobilized by forming an amide bond between them.
Since the pore size depends on the gel monomer concentration used, it is necessary to set the concentration low, which makes it difficult to form a gel, but according to the present invention, as described above, the monomer concentration is low. Since both gels can be formed and the pore size can be increased, proteins with a large molecular size, such as antibodies, can diffuse into the gel and interact with the immobilized protein. Detection is also possible.

  Thus, when investigating an interaction with a biomolecule, first, the produced protein array substrate is irradiated with ultraviolet light, and the intensity of transmitted light is measured. Then, after adding the sample solution containing the target biomolecule to the entire protein array substrate, the nonspecifically adsorbed substance is washed away, leaving only the biomolecule adsorbed by the specific interaction, and again. Irradiate with ultraviolet light, measure the intensity of transmitted light, and compare with that before adding the sample solution. A spot where the intensity of transmitted light is reduced indicates that the biomolecules in the sample are adsorbed to the immobilized protein of the spot and absorbs the irradiation light. The degree of decrease indicates the amount of the adsorbed biomolecules. Reflects. In this way, the interaction between the immobilized protein and the substance in the sample can be detected and analyzed.

  Examples of the present invention are shown below, but the present invention is not limited to the examples.

Example 1
Production of protein array substrate In production of a protein array substrate, first, bind silane, which is a silane coupler capable of bonding glass and acrylamide, is introduced into the surface of quartz glass, and then the silicon film frame is made of glass. Installed on the surface, and then poured an acrylamide solution containing an amino group-containing polymer into the frame, covered with a water-repellent cover glass, and polymerized in a petri dish in a steam atmosphere for about 16 hours, thereby forming a protein microarray A gel substrate was prepared. Details of each process are described below.
Bind silane (purchased from GE Healthcare) can form a covalent bond with both quartz glass (OPSQ-40S03-P, purchased from Sigma Kogyo) and acrylamide molecule, allowing them to be firmly bound together. In this way, it was introduced on the surface of quartz glass. Prepare a solution by mixing 800 μl of ethanol, 20 μl of acetic acid, 180 μl of pure water, and 1 μl of bound silane solution, add 100 μl of this solution to the entire surface of the quartz glass in a draft chamber, and use a Kimwipe Wipe off the entire surface and dry for 1-2 hours.

A silicon film having a thickness of 0.3 mm (purchased from ASONE) was cut into a size of 30 mm × 30 mm, and an area of 20 mm × 20 mm at the center was cut out with a cutter knife. The frame body produced in this manner was placed on a 40 mm × 40 mm quartz glass, and lightly rubbed from above to adhere.
Acrylamide (purchased from Bio-Rad) and N, N'-diallyl-L-tartaric acid diamide (DATD, purchased from Wako Pure Chemical Industries) as a cross-linking agent are dissolved in pure water, and the concentration is 10% and the cross-linking agent content is 15%. A monomer solution was prepared. To 100 μl of this solution, add poly-L-lysine (poly-L-lysine with a molecular weight of 30,000-70,000, purchased from Sigma) and pure water, and finally the polyacrylamide concentration is 5%, the cross-linking agent content is 15%, poly-L-lysine 200 μl of a solution having a concentration of 0.4% was prepared. Next, 0.2 μl of TEMED as a polymerization accelerator and 2 μl of 10% ammonium persulfate solution were added and mixed well, and then 150 μl of this solution was poured into a frame on quartz glass. Immediately after that, a water-repellent cover glass (24X24mm Thickness 0.12-0.17mm, purchased from Matsunami Glass) was covered so that air bubbles would not enter. The water repellent treatment of the cover glass was performed as follows. Immerse the cover glass in 5 ml of ripipylsilane solution (purchased from GE Healthcare), a water-repellent silane solution, leave it for about 1 minute in a draft chamber, and then use Kimwipe to remove excess surface. Silane was wiped off.

After pouring the acrylamide solution into the frame on the quartz plate and covering it with a water-repellent cover glass, put it in a petri dish containing a Kimwipe containing 2 ml of pure water in advance and seal it. In a steam atmosphere. The petri dish was placed in a constant temperature incubator set at 25 ° C. for about 16 hours to polymerize acrylamide.
After the polymerization is complete, use a transilluminator to irradiate the entire quartz plate with UV light for about 5 minutes, then remove the cover glass using tweezers, remove the silicon rubber frame, and then remove the pure silica in the petri dish. It was immersed in water and stirred for about 30 minutes. Thereafter, the pure water was changed three times, and the polymerized gel substrate was washed by stirring for a total of about 2 hours to remove unpolymerized monomers and the like. After that, put the whole quartz plate in a petri dish containing 2 ml of pure water in advance and place it in a steam atmosphere by sealing and put it in an incubator set at 35 ° C for about 16 hours. Thus, the moisture on the surface was removed without drying the gel substrate, so that the protein for immobilization could be spotted.

Example 2
Immobilization of the fluorescent protein for immobilization on the substrate for the array As the fluorescent protein for immobilization, a protein obtained by binding the amino acid sequence for immobilization to the carboxy terminal side of the green fluorescent protein was used. A gene encoding this amino acid sequence was artificially synthesized, incorporated into an expression vector (pUC18, purchased from Toyobo), expressed in E. coli, and purified to prepare the target protein. If a person skilled in the art can obtain a gene encoding a fluorescent protein, the target protein can be easily prepared by the same method.
Dissolve the fluorescent protein for immobilization prepared in this way in 10 mM phosphate buffer (pH 7.0) so that the final concentration is 4 mg / ml, and add 60 nl each to 9 points in a 3X3 lattice at 1 mm intervals I spotted it. A spot device (purchased from Musashi Engineering) was used for the spot. Immediately after spotting, the protein solution formed approximately 0.4 mm droplets on the surface, but then quickly absorbed into the gel.

  The substrate on which the protein was spotted as described above was immersed in a 10 mM phosphate buffer (pH 7.0) containing 1 mM DTT for about 1 hour at 37 ° C. to carry out a protein reduction reaction. After washing in a petri dish with 10 mM phosphate buffer (pH 7.0), it was immersed in 10 mM phosphate buffer (pH 7.0) containing 5 mM 2-nitro-5-thiocyanobenzoic acid (NTCB). The mixture was allowed to stand at room temperature for about 4 hours to carry out a cyanation reaction. Then, the immobilization reaction was performed by immersing in a 10 mM borate buffer (pH 9.5) in a petri dish and gently stirring at room temperature for about 24 hours. After completion of the immobilization reaction, unreacted proteins and side reaction products were removed by immersing in a 10 mM phosphate buffer (pH 7.0) containing 1 M KCl in a petri dish and gently stirring for about 2 hours.

Example 3
Detection of immobilized green fluorescent protein by optical means The fluorescent protein array prepared as described above was set in a dedicated cell and filled with 10 mM phosphate buffer (pH 7.0). This cell was irradiated with 280nm UV light separated by a spectroscope (purchased from Shimadzu Corporation) using a dedicated xenon lamp as the light source, and the light transmitted through the protein array in the cell was photographed with an ultraviolet CCD camera ( FIG. 5).
According to this, since the 280 nm ultraviolet light transmitted in each spot decreased and appeared black, it was shown that the absorption of 280 nm light by the immobilized fluorescent protein could be detected. In addition, it can be seen that the degree of decrease in transmitted light in each spot or spot in the protein array is almost uniform, and the fluorescent protein is uniformly immobilized on the array substrate.

Example 4
Immobilization of antibody-binding protein for immobilization on array substrate As antibody-binding protein for immobilization, an amino acid for immobilization on the carboxy-terminal side of a sequence in which two antibody-binding domains of protein A derived from Staphylococcus aureus are linked What combined the arrangement | sequence was used. A gene encoding this amino acid sequence was artificially synthesized, incorporated into an expression vector (pUC18, purchased from Toyobo), expressed in E. coli, and purified to prepare the target protein.

The dried preparation of the antibody-binding protein for immobilization thus prepared was dissolved in 10 mM phosphate buffer (pH 7.0) so that the final concentration was 10 mg / ml. Spotted 9 points at 1.2mm intervals. Thereafter, an immobilization reaction was performed by the method shown in Example 2, and then the unreacted amino group in the gel substrate was acetylated to lose the charge as follows. Immerse the substrate in 1 ml of 0.5 M borate buffer (pH 9.5), add 10 μl acetic anhydride 6 times approximately every 10 minutes while stirring at room temperature, and then 3 times with 10 mM phosphate buffer (pH 7.0). The above was washed.
The thus-prepared antibody-immobilized protein array can be immersed in a 10 mM phosphate buffer (pH 7.0) and stored at 4 ° C.

Example 5
Antibody molecule addition and detection of adsorbed antibody by optical means The antibody-bound protein array prepared as described above was set in a dedicated cell and filled with 10 mM phosphate buffer (pH 7.0). This cell is irradiated with 280 nm UV light that is split by a spectroscope (purchased from Shimadzu Corporation) using a dedicated xenon lamp as the light source, and the light transmitted from the antibody-binding protein array in the cell is photographed with an ultraviolet CCD camera. (FIG. 6A). At this stage, no spots are observed. This is because the immobilized antibody-binding protein contains only two amino acid residues that absorb ultraviolet light at 280 nm, and therefore has almost no light absorption.
Thereafter, antibody molecules were added to the protein array as described below, and the adsorbed antibody molecules were detected by optical means. Antibody molecules (I4506, purchased from Sigma) are dissolved in 10 mM phosphate buffer (pH 7.0) to a final concentration of 5 μg / μl, and the antibody-bound protein array is immersed in 1.5 ml of this antibody solution for about 16 hours. Gently infiltrated. After that, by immersing in 10 mM phosphate buffer (pH 7.0) containing 1M KCl and gently stirring for about 2 hours, leaving only the antibody molecules adsorbed by the specific interaction, the non-specifically adsorbed antibody The molecule was removed. Thereafter, this antibody-bound protein array was set again in a dedicated cell, irradiated with 280 nm ultraviolet light, and the ultraviolet light transmitted from the protein array was photographed with a CCD camera (FIG. 6B). It has been observed that the transmitted light at each spot decreases after addition of antibody molecules and appears black. This is because the adsorbed antibody molecules absorb UV light at 280 nm. It was shown that the state of interacting with the binding protein and adsorbing could be detected without labeling.

Diagram showing the conventional method for producing protein arrays The figure which shows the usage aspect of the apparatus for protein array board | substrate preparation to this invention The figure which shows typically the superposition | polymerization process of the gel monomer in the preparation process of the protein array substrate in this invention. The figure which shows typically the water | moisture-content removal process in the preparation process of the protein array substrate in this invention. A photograph in which green fluorescent protein is immobilized on the protein array substrate of the present invention and ultraviolet light is irradiated to photograph transmitted light The photograph which shows that the mode that an antibody couple | bonds with the protein array of the invention which fixed the antibody binding protein was detected by the change of the transmitted light before and behind addition of an antibody. A: Before antibody addition, B: After antibody addition

Claims (10)

  A transparent substrate, a frame body placed on the substrate and containing a gel monomer-containing solution, and a cover member that covers the upper portion of the frame body. An apparatus for preparing a protein array substrate, wherein a space for polymerizing and gelling gel monomers is formed in a state where the gel monomers are blocked from the substrate.   The apparatus for producing a protein array substrate according to claim 1, wherein the frame is formed of a material having adhesion to a transparent substrate.   The apparatus for producing a protein array substrate according to claim 2, wherein the frame is made of silicon rubber or silicon film.   The apparatus for producing a protein array substrate according to any one of claims 1 to 3, wherein the cover member is a water-repellent flat plate.   The apparatus for producing a protein array substrate according to claim 5, wherein the gel monomer-containing solution contains acrylamide, a crosslinking agent thereof, an amino group-containing polymer, and a polymerization accelerator.   A frame is placed on a transparent substrate, a gel monomer-containing solution is allowed to flow into the frame, and then the gel monomer is polymerized and gelled in a state of covering the frame with a cover member and blocking from the outside air. A method for producing a protein array substrate.   The method for producing a protein array substrate according to claim 6, wherein the gel monomer-containing solution contains acrylamide, a crosslinking agent thereof, an amino group-containing polymer, and a polymerization accelerator.   The method for producing a protein array substrate according to claim 6 or 7, wherein the polymerization of the gel monomer is performed under a constant temperature condition in a water vapor atmosphere.   9. The method for producing a protein array substrate according to claim 8, wherein the gel monomer is washed with water after polymerization, and further, moisture on the surface of the gel substrate is removed in a container under a water vapor atmosphere under a constant temperature condition.   A protein array, wherein a protein is immobilized on a protein array substrate produced by the production method according to claim 6.