JP2007024831A - Blocking method for array - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an array capable of attaining array analysis capable of restraining nonspecific adsorption of a biomolecule, and capable of obtaining a data of high reliability, in particular, when used in surface plasmon imaging measurement. <P>SOLUTION: In this blocking method for the array, a solution containing an HSP70 family protein from which an ATPase domain is removed is made to act on the array immobilized with the biomolecule. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an array blocking method capable of suppressing nonspecific adsorption of biomolecules by blocking “HSP70 family proteins” in a substance interaction analysis system using a peptide chip.

  In recent years, biochips used for biomolecule interaction analysis, profiling of expressed molecules, or diagnosis have attracted attention. The operation is facilitated by immobilizing the biomolecule on the substrate, and in some cases, the interaction of a very large number of substances can be analyzed. In particular, peptide arrays in which peptides with relatively small molecular weights are immobilized on a substrate have relatively few problems of denaturation such as proteins, and have strong combinatorial chemistry aspects. It has come to be widely used for searching and the like.

  However, it is the influence of nonspecific adsorption that becomes a problem when observing the interaction. Non-specific adsorption refers to a case where the target substance adsorbs non-specifically to molecules that do not interact with each other. As a result, a false positive determination is given, which is not preferable. In order to suppress nonspecific adsorption, biochips in which hydrophilic polymers such as dextran and polyethylene glycol (PEG) are immobilized on the surface have been developed. These hydrophilic polymers are known to have an effect of suppressing nonspecific adsorption of biomolecules.

  A biosensor in which non-specific adsorption is suppressed by forming a hydrophilic polymer gel matrix on the biosensor surface is shown. In this method, functional groups are introduced into the gel matrix, and biomolecules are immobilized by covalent bonds using the functional groups. Specifically, the carboxyl group of carboxymethyl dextran was activated with water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the formed succinimide group and the biomolecule were successfully immobilized (Non-patent Document). 1). However, since the formed succinimide group does not react 100%, it is necessary to block the remaining succinimide. Here, succinimide groups are blocked using ethanolamine, and a technique using a hydrophilic polymer as a blocking material is not shown.

  A means for reacting a hydrophilic polymer on a substrate after immobilizing a biomolecule is known (Patent Document 1). However, here, the maleimide group on the chip is reacted with the thiol group of the target molecule to immobilize the target molecule on the chip, but the subsequent step of blocking the maleimide group is not included. For this reason, the maleimide group remains and there is a concern of causing nonspecific adsorption problems. Further, since a hydrophilic polymer that can react with the immobilized biomolecule is used, the biomolecule may be modified and denatured.

  In addition, as a blocking agent used in conventional immunoassays and the like, many proteins directly extracted from biological components have been used. In particular, bovine-derived albumin and casein have been widely used. However, in recent years, various restrictions have been added due to problems such as mad cow disease. On the other hand, methods for producing them using recombinants have the advantage that pathogens (substances) can be eliminated, but few have come to practical use because of problems such as productivity.

Therefore, it can be said that an attempt to use a protein derived from Escherichia coli as an alternative protein is preferable from the standpoint of productivity. However, proteins that can be expressed in large quantities do not necessarily have good blocking efficiency, and there is a problem that a protein with excellent blocking efficiency that can be produced by a recombinant must be found.
In recent years, the gene sequences of various organisms have been clarified, but the law of finding a protein with better blocking efficiency than the predicted amino acid sequence is not yet known, and it takes a lot of trouble to find such a protein. It was thought to be a work to be done. For these reasons, there has been a demand for an alternative protein with excellent blocking efficiency that can be mass-produced with a recombinant.

Anal. Biochem. 198, 268, 1991 US Pat. No. 6,127,129

  An object of the present invention is to provide a biochip blocking technique for suppressing nonspecific adsorption of biomolecules. As a result, there is a need to obtain a blocking means that can accurately evaluate an interaction when used for analyzing an interaction with a biomolecule immobilized on a substrate.

  As a result of intensive studies, the present inventor has found that the above problems can be solved by the following means, and has reached the present invention.

That is, this invention consists of the following structures.
1. A method for blocking an array, wherein a solution containing an HSP70 family protein from which an ATPase domain has been removed is allowed to act on an array on which a biomolecule is immobilized.
2. A method for blocking one array, wherein the “HSP70 family protein” from which the ATPase domain has been removed is selected from DnaK, Ssa1p, Ssc1p, Kar2p, HSP70, Bip, mHsp70 and HSC70.
3. A method for blocking one or two arrays, wherein the substrate binding domain of “HSP70 family protein” is used.
4). A method for blocking an array according to any one of 1 to 3, wherein a protein derived from DnaK protein is used.
5. A method for blocking an array according to any one of 1 to 4, wherein a protein obtained by removing a part of the amino acid sequence of the DnaK protein is used.
6). Any one of 1 to 5, wherein a protein from which a part of the amino acid sequence of DnaK protein has been removed, wherein at least the amino acid sequence from the N-terminal to the 387th and at most the 472th amino acid sequence has been removed is used Array blocking method.
7). Any one of 1 to 6, wherein a protein from which a part of the amino acid sequence of DnaK protein has been removed, wherein at least the amino acid sequence from the N-terminal to the 387th and at most the 418th amino acid sequence has been removed is used Array blocking method.
8). A method for blocking an array according to any one of 1 to 7, wherein a protein comprising an amino acid sequence from the 419th to 607th amino acids of the DnaK protein is used.
9. The method for blocking an array according to any one of 1 to 8, wherein a protein in which a hydrophilic amino acid in a part of the DnaK protein from which the ATPase domain or a part thereof has been removed is substituted with a hydrophobic amino acid is used.
10. A protein obtained by removing a part of the amino acid sequence of the DnaK protein from which the ATPase domain or part thereof has been removed, wherein the aspartic acid of amino acid numbers 479 and 481 is substituted with valine. Any array blocking method.
11. A method for blocking an array according to any one of 1 to 10, wherein a protein comprising the amino acid sequence of 384 to 607 of the DnaK protein and having aspartic acid at amino acid numbers 479 and 481 substituted with valine is used.
12 The array blocking method according to any one of 1 to 11, wherein the array surface is gold.
13. The method for blocking an array according to any one of 1 to 12, wherein the substrate is a transparent substrate.
14 The method for blocking an array according to any one of 1 to 13, wherein the substrate is glass.
15. The array blocking method according to any one of 1 to 14, wherein the array is used for surface plasmon resonance (SPR) analysis.

  By adopting the array blocking method of the present invention, a highly accurate measurement system using an array in which nonspecific adsorption is suppressed can be obtained.

  The present invention relates to an array blocking method using a so-called “HSP70 family protein” composed of DnaK, Ssa1p, Ssc1p, Kar2p, HSP70, Bip, mHsp70 and HSC70, which are known as molecular chaperones.

  Among them, the substrate binding domain of HSP70 (DnaK), which is a kind of E. coli heat shock protein, is preferably used. This protein is composed of 638 amino acids, and consists of an “ATPase (ATP binding) domain” composed of amino acids 1 to 385 and a “substrate binding domain” composed of amino acids 386 to 638 (FIG. 1). ). SEQ ID NO: 1 shows the sequence of the DnaK protein. The structure of this protein (DnaK384-607) has already been revealed by NMR analysis, and two structural regions (domains), that is, an N-terminal β-sheet region (domain) and a C-terminal α-helix. It is known that it consists of regions (domains). In addition, the hydrophilicity / hydrophobicity ratio on the N-terminal side of the sequence was 0.5, 0.89 on the C-terminal side, and the difference was as high as 0.39 by calculation. This protein (DnaK384-607) is useful for blocking.

  In the present invention, proteins derived from HSP70 family proteins are preferably used, and proteins derived from DnaK protein of E. coli are more preferably used. Preferably, the substrate binding domain of the HSP70 family protein from which the ATPase domain has been removed is preferably used, and more preferably the substrate binding domain of the DnaK protein from which the ATPase domain of E. coli has been removed.

  The protein belonging to the HSP70 family used in the present invention is not particularly specified, but DnaK of E. coli, Ssa1p present in yeast cytoplasm, Ssc1p present in yeast mitochondria, Kar2p present in yeast endoplasmic reticulum, HSP70 present in mammalian cytoplasm. , Bip present in mammalian endoplasmic reticulum, mHsp70 present in mammalian mitochondria, and HSC70, which is a homologue of HSP70 that is constantly expressed regardless of the presence or absence of heat shock. Many homologues are known in the HSP70 family, and the above are some of them, and it can be easily predicted that similar effects can be expected from homologues other than those listed above.

  Moreover, a protein having a more excellent blocking efficiency can be obtained by adding a mutation to the β sheet structure portion of DnaK to improve the hydrophobicity of the β sheet portion. That is, (1) removing a part of the N-terminal of the β sheet to expose a more hydrophobic portion of the β sheet (destroying the β sheet structure to improve hydrophobicity), (2) β sheet The above hydrophilic amino acid is replaced with a hydrophobic amino acid. As a result, DnaK419-607 from which the N-terminal portion of the β-sheet structure was removed and DnaK384-607 (D479V, D481V) in which the hydrophilic amino acids on the β-sheet were replaced with hydrophobic amino acids were found to have a significant improvement in blocking efficiency. Can do. In particular, a significant improvement in blocking efficiency can be observed in DnaK 419-607. That is, it is considered that the change in the structure of the hydrophobic domain causes the change in the structure of the hydrophobic domain and the improvement in the hydrophobicity of the hydrophobic domain leads to an improvement in the blocking ability.

  In the present invention, a DnaK protein having improved blocking efficiency, characterized by removing amino acid sequences from at least the N-terminal to the 387th and at most the 472th, is more preferable. Further, the present invention is preferably a DnaK protein with improved blocking efficiency, characterized in that the amino acid sequence from at least the N-terminal to the 387th and at most the 418th amino acid sequence is removed. Particularly preferred is a protein consisting of the amino acid sequence from the 419th to 607th amino acid sequence of the DnaK protein.

  In the present invention, a protein obtained by substituting a part of the hydrophilic amino acid of the DnaK protein from which the ATPase domain or part thereof is removed with a hydrophobic amino acid may be used. More specifically, it is a protein from which a part of the amino acid sequence of the DnaK protein from which the ATPase domain or part thereof has been removed has been removed, wherein the aspartic acid at amino acid numbers 479 and 481 has been replaced with valine, thereby improving the blocking efficiency. . More preferably, a protein comprising the amino acid sequence of the 384th to 607th DnaK protein and having aspartic acid at amino acid numbers 479 and 481 substituted with valine is used.

  The protein or protein fragment used in the present invention may have a tag such as a histidine tag as long as the effects of the present invention are not impaired. Furthermore, a tag that is not generally known or an arbitrary amino acid sequence may be added as long as the effects of the present invention are not impaired.

  Although the expression method of this protein is not specifically limited, The method of expressing using a prokaryote is preferable, Furthermore, the method of expressing using Escherichia coli is further more preferable. The expression vector is not particularly limited as long as it is generally used for expression.

  Proteins belonging to the HSP70 family used in the present invention are not particularly limited, but include DnaK of E. coli, Ssa1p present in yeast cytoplasm, Ssc1p present in yeast mitochondria, Kar2p present in yeast endoplasmic reticulum, and HSP70 present in mammalian cytoplasm. Bip present in the mammalian endoplasmic reticulum, mHsp70 present in mammalian mitochondria, and HSC70, which is a homologue of HSP70 that is constantly expressed regardless of the presence or absence of heat shock. Numerous homologs are known in the HSP70 family, and those listed above are some of them, and may be homologs other than those listed above.

  The HSP70 fragment used in the present invention is characterized in that the ATPase domain is removed. However, as long as sufficient blocking activity is exhibited, the entire region of the ATPase domain may not be removed. For example, the ATPase domain refers to the 1-383 region on the C-terminal side in DnaK, and a part of the ATPase domain is removed to such an extent that the ATPase activity is lost. For example, in DnaK, the 384-638 fragment can also be used.

  The blocking method in the present invention can be applied to various applications, and is particularly great for optical detection methods such as surface plasmon resonance (SPR), sum frequency generation (SFG), localized plasmon resonance (LPR), and ellipsometry. Demonstrate the effect. Among these, it is particularly useful in an analysis system using SPR. In a general chip or array, detection by a labeling means using a fluorescent substance or a radioisotope is used as a method for detecting an interaction. In this case, it is possible to detect only whether or not the label substance is finally bound, and even if a substance not related to the interaction is adsorbed non-specifically, it is not erroneously detected. Therefore, if the interacting target substance is not non-specifically adsorbed to the negative control, it can be determined that the measurement is accurate.

  In SPR, SFG, LPR, and ellipsometry, a metal substrate is used. In the present invention, the material of the substrate is preferably gold which is very stable in acid, alkali, organic solvent and the like. In fact, gold is a material frequently used in the above optical detection method. Further, the material supporting gold is preferably transparent, and more preferably transparent glass. This is because transparent glass is not only easily available, but also very suitable for measuring SPR and LPR.

    As a method of forming the metal substrate, a method of coating a thin metal layer is preferable. A method for coating the metal is not particularly limited, but generally, a vapor deposition method, a sputtering method, an ion coating method, or the like is selected. In order to provide an optical detection method, it is necessary to control the thickness of the thin metal layer at the nano level. The thickness of the thin metal layer is not particularly limited, but is generally selected in the range of 30 nm to 80 nm. In order to suppress peeling of the thin metal layer, a chromium layer or a titanium layer of 0.5 nm to 10 nm may be coated on the substrate in advance.

  The SPR imaging method using this SPR is a method of monitoring the state of interaction between substances by irradiating a polarized light beam over a wide range and analyzing the reflected image by making full use of image processing technology or the like. Yes, it is possible to screen a chip on which a plurality of substances are immobilized, and to observe the morphology of an object adsorbed on the surface with high sensitivity. In the SPR imaging method, in order to analyze the reflected image, a means for irradiating the chip with a polarized light beam in a wide range and sufficiently securing the illuminance of the light beam is required. An example is shown in FIG. The brighter the illuminance of the polarized light flux, the more preferable is the sensitivity of the sensor.

  The type of the light source is not particularly limited, but it is preferable to use light including near infrared light that makes the change in the SPR resonance angle particularly sensitive. Specifically, a white light source capable of irradiating light over a wide range such as a metal halide lamp, a mercury lamp, a xenon lamp, a halogen lamp, a fluorescent lamp, and an incandescent lamp can be used. A halogen lamp that is sufficiently high, has a simple light power supply and is inexpensive is particularly preferable.

  A normal white light source has a defect that light and dark unevenness occurs in the filament portion. If the light from the light source is irradiated as it is, light and dark unevenness occurs in the image obtained by reflection, and it becomes difficult to evaluate screening and morphological changes. Therefore, as a means for uniformly irradiating the chip with light, a method of making the light into parallel light after passing through the pinhole is preferable. The means for passing a pinhole is preferable as a means for obtaining a light beam having a uniform brightness, but there is a drawback that the illuminance decreases when light is passed through the pinhole as it is. Therefore, as a means for ensuring sufficient illuminance, it is preferable to use a method in which a convex lens is installed between the pinhole and the light source, and condensed to pass through the pinhole.

Since the white light source is radiated light, it needs to be converted into parallel light using a convex lens before condensing. Parallel light can be obtained by installing a light source near the focal length of the convex lens. By installing another convex lens and installing a pinhole in the vicinity of the focal length of the lens, it is possible to pass the condensed light through the pinhole. The light that intersects and passes through the pinhole is converted into parallel light by the CCTV lens for the camera. The cross-sectional area of the parallel light beam obtained at this time is preferably adjusted to 10 to 1000 mm ² . This method enables a wide range of screening and morphological observation.

  When the interaction is monitored, the polarized light beam is applied to the opposite surface of the metal thin film on which the substance or the substance aggregate is fixed. The polarized light beam is applied to the opposite surface of the metal thin film on which the substance or substance aggregate is fixed, and the reflected light beam is obtained. The reflected light beam from the metal thin film passes through a near-infrared wavelength optical interference filter and transmits only light in the vicinity of a certain wavelength before being photographed by a CCD camera.

  The center wavelength of the optical interference filter is preferably 600 to 1000 nm with high SPR sensitivity. The wavelength width of the wavelength at which the transmittance of the optical interference filter is half that at the maximum is called a half-value width, but the smaller the half-value width, the sharper the wavelength distribution is, and more specifically, the half-value width is preferably 100 nm or less. An image photographed by the CCD camera through the optical interference filter is taken into a computer, and a change in brightness of a certain part can be evaluated in real time, and an entire image can be evaluated by image processing. Thus, it is possible to screen a chip on which a plurality of substances are immobilized, and to observe the morphology of an object adsorbed on the surface with high sensitivity.

  The chip for SPR used in the present invention preferably comprises a metal substrate in which a metal thin film is formed on a transparent substrate, and directly or indirectly, chemically or physically on the metal thin film, a substance or a substance. A slide on which the assembly is fixed is used. The material of the substrate is not particularly limited, but it is preferable to use a transparent material. Specific examples include glass or plastics such as polyethylene terephthalate (PET), polycarbonate, and acrylic. Of these, glass is particularly preferable.

The thickness of the substrate is preferably about 0.1 to 20 mm, and more preferably about 1 to 2 mm. In order to achieve the purpose of evaluating the reflection image from the metal thin film, the SPR resonance angle is as small as possible, and the captured image is not likely to be distorted and is easy to analyze. Therefore, the refractive index n _D of the transparent substrate or the transparent substrate and the prism in contact with the transparent substrate is preferably 1.5 or more.

  The biomolecule immobilized on the array used in the present invention is not particularly limited, and all substances generally included in the category of biological constituents are included. Specifically, protein, peptide, DNA, RNA, sugar, lipid and the like are exemplified. Further, it is useful not only in the SPR array as described above but also in an array used for detection by fluorescence or radioisotope. In this case, the array surface does not necessarily have to be gold.

  In the present invention, the concentration of the solution containing the HSP70 family protein from which the ATPase domain has been removed is preferably about 0.1 to 20 mg / ml, preferably 1 to 10 mg / ml, more preferably 2 to 10 mg / ml. The lysis solution is not particularly limited, but a neutral buffer solution is preferable. The pH is in the range of 6-8, preferably 6.5-7.5. The type of the buffer solution is not particularly limited, and various types such as a phosphate buffer solution, a Tris buffer solution, and a HEPES buffer solution are applicable.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not particularly limited to the examples.

[Example 1]
(Array production)
_PEG 3 -OH alkanethiol terminal functional groups is a thiol group; ethanol 7 ml (. Ltd. SensoPath hereinafter referred PEG thiol) at a concentration of 1 mM: water = 6: was dissolved in a mixed solution of 1. The terminal thiol group exhibits particularly excellent metal binding to gold. A gold-deposited slide in which 3 nm of chromium was vapor-deposited on an SF15 glass slide of 18 mm square and 2 mm thick and 45 nm of gold was vapor-deposited was immersed in the PEG thiol solution for 3 hours to bond PEG thiol to the entire gold substrate.

  A photomask was placed on the slide and irradiated with a 500 W ultra-high pressure mercury lamp (manufactured by USHIO) for 2 hours to remove PEG thiol in the UV irradiation part. The photomask has 96 square holes of 500 μm square (consisting of 8 × 12 patterns), and the pitch between the hole centers is designed to be 1 mm. UV light is transmitted through the holed portion of the photomask, and the slide is irradiated and patterned. PEG remains in the unirradiated portion, and functions as a reference portion as a background portion of the chip.

It was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 1 hour to form a self-organized surface of 8-AOT on the UV irradiation part. After washing with PBS and water, a linker having reactivity with an amino group whose end was modified with biotin was allowed to act. Two types of linkers, Biotin-AC ₅ Sulfo-Osu (manufactured by Dojin Chemical) and NHS-dPEG ₄ -OSu (manufactured by Quanta Biodesign), were used. The respective chemical structures are shown in formulas (I) and (II). Hereinafter, Formula (I) is shown as AC5 and Formula (II) is shown as PEG.

  A solution in which both AC5 and PEG were dissolved in PBS to a concentration of 1 mM was prepared. Moreover, the solution which mixed each equal amount was also prepared. These three types of linker solutions were spotted 10 nl at a time as shown in FIG. 3, using a MultiSPRinter automatic spotter (manufactured by Toyobo). Thereafter, it was reacted with 8-AOT on the gold surface at room temperature for 3 hours in a wet environment. In this way, biotin could be introduced onto the gold surface.

  The biotin surface chip was washed with PBS and water, and then immobilized with streptavidin. Streptavidin was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl, pH 7.2) at 1 mg / ml, and the MultiSPRinter automated test was performed on the 1, 2 and 3 spots in FIG. Spotting was performed 10 nl at a time using a potter (manufactured by Toyobo). Then, the immobilization reaction was performed by leaving still for 30 minutes at room temperature in a wet environment. Thus, streptavidin could be immobilized on the surface.

(Blocking process)
After washing the surface with immobilized streptavidin with PBS and water, DnaK (419-607) was dissolved in a phosphate buffer (20 mM phosphate, 150 mM NaCl, pH 7.2) at a concentration of 2 mg / ml, 300 μl was poured onto the chip and reacted at room temperature for 1 hour.

(Measurement by SPR imaging method)
The peptide-immobilized chip thus obtained was set in an SPR imaging device (MultiSPRinter: manufactured by Toyobo), and phosphate buffer (20 mM phosphate, 150 mM NaCl, pH 7.2) was used as a running buffer at a rate of 100 μl / min. In the flow cell. After confirming that the signal from the SPR was stabilized, an anti-streptavidin antibody (manufactured by Vector) was injected into the cell in the SPR device to act. The antibody was used by adjusting the concentration to 10 μg / ml with the above running buffer. When the signal rise reached a plateau state, the running buffer was fed again for washing. The change of the SPR signal at that time was observed. In addition to the spot site of each substrate, the signal change was also observed in the background.

  After the above measurement, analysis by SPR imaging was also examined. At the time of the SPR analysis, an image obtained by imaging the surface of the array with a CCD camera is taken every 2 seconds, and from the image taken at the time after the antibody reaction, the image at the time before the reaction is obtained from the image calculation processing software Scion Image By performing a subtraction process using (made by Scion Corp.), it is possible to show how the antibodies in the entire array are bound.

(Observation results and discussion)
The results are shown in FIG. (A) shows the SPR sensorgram, and (B) shows the result of SPR imaging. In any of the linkers, a sharp antibody binding signal is confirmed in the spots 1, 2, and 3 in FIG. 3 where streptavidin is immobilized. On the other hand, almost no antibody binding was observed in the blank spots 4, 5, and 6 in FIG. 3 in which streptavidin was not immobilized, and a result with excellent specificity was obtained.

[Comparative Example 1]
(Array production)
The same operation as in Example 1 was performed.
(Blocking process)
Here, the blocking operation was not performed, and the process proceeded directly to the next step (measurement by SPR imaging method).
The same operation as in Example 1 was performed.

(Observation results and discussion)
The results are shown in FIG. (A) shows the SPR sensorgram, and (B) shows the result of SPR imaging. In this case, not only the spots 1, 2, and 3 in FIG. 3 where streptavidin is immobilized but also the blank spots 4, 5, and 6 in FIG. 3 where streptavidin is not immobilized are not specific. Antibody adsorption is observed. In particular, the tendency is remarkable when AC5 is used. An increase in signal is also observed in the background portion. From the above results, it was possible to confirm the excellent effect of the blocking treatment in the present invention.

  Non-specific adsorption is effectively suppressed in the array treated by the blocking method of the present invention, and accurate measurement is possible. The method is also easy and is expected to make a significant contribution to industry.

It is a figure which shows the outline | summary of colon_bacillus | E._coli DnaK protein. It is a figure which shows the principle of SPR imaging. FIG. 3 is a diagram showing a linker arrangement pattern using the SPR array produced in Example 1. It is a figure which shows the analysis result of SPR imaging in Example 1 (A: sensorgram, B: imaging result). It is a figure which shows the analysis result of SPR imaging in the comparative example 1 (A: sensorgram, B: imaging result).

Claims (15)

  A method for blocking an array, wherein a solution containing an HSP70 family protein from which an ATPase domain has been removed is allowed to act on an array on which a biomolecule is immobilized.   The method for blocking an array according to claim 1, wherein the "HSP70 family protein" from which the ATPase domain has been removed is selected from DnaK, Ssa1p, Ssc1p, Kar2p, HSP70, Bip, mHsp70 and HSC70.   The method for blocking an array according to claim 1 or 2, wherein a substrate binding domain of "HSP70 family protein" is used.   4. The array blocking method according to claim 1, wherein a protein derived from DnaK protein is used.   The method for blocking an array according to any one of claims 1 to 4, wherein a protein obtained by removing a part of the amino acid sequence of the DnaK protein is used.   6. A protein from which a part of the amino acid sequence of the DnaK protein has been removed, wherein at least the amino acid sequence from the N-terminal to the 387th to the 472st is removed. A method for blocking an array according to claim 1.   7. A protein from which a part of the amino acid sequence of the DnaK protein has been removed, wherein at least the amino acid sequence from the N-terminal to the 387th and at most the 418th amino acid sequence is used. A method for blocking an array according to claim 1.   The method for blocking an array according to any one of claims 1 to 7, wherein a protein comprising an amino acid sequence from 419 to 607 of the DnaK protein is used.   The method for blocking an array according to any one of claims 1 to 8, wherein a protein obtained by substituting a part of hydrophilic amino acid of DnaK protein from which ATPase domain or a part thereof is removed with a hydrophobic amino acid is used.   A protein obtained by removing a part of the amino acid sequence of the DnaK protein from which the ATPase domain or a part thereof has been removed, wherein the aspartic acid of amino acid numbers 479 and 481 is substituted with valine. 10. The method for blocking an array according to any one of 9 above.   The method for blocking an array according to any one of claims 1 to 10, wherein a protein comprising the amino acid sequence of 384 to 607 of the DnaK protein, wherein the aspartic acid of amino acid numbers 479 and 481 is substituted with valine, is used.   The array blocking method according to claim 1, wherein the array surface is gold.   The array blocking method according to claim 1, wherein the substrate is a transparent substrate.   14. The array blocking method according to claim 1, wherein the substrate is made of glass.   The array blocking method according to claim 1, wherein the array is used for surface plasmon resonance (SPR) analysis.