JP2008309617A - Protein crystalizing device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a weighing means capable of easily weighing the dispensed amount of a protein-containing sample. <P>SOLUTION: The means for weighing the dispensed amount of the protein-containing sample has two or more weighing recesses different in volume. The weighing recesses are preferably arranged in order of volume. The volume of each weighing recess is preferably within a range from 0.001 to 2 μl. Further preferably, the shape of the weighing recesses is hemispherical or cylindrical. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an apparatus and method for crystallization of proteins or screening for crystallization conditions.

  In recent years, research called structural genomics has been conducted in an attempt to elucidate the function of a gene that encodes a protein by clarifying the relationship between the three-dimensional structure of the protein and the function of the protein.

  The analysis of the three-dimensional structure of a protein usually involves screening the crystallization conditions of the protein to be analyzed, performing crystallization under optimal crystallization conditions, and then subjecting the obtained crystals to X-ray structural analysis. Has been done. Here, in the process of screening protein crystallization conditions, a lot of time and a large amount of protein-containing samples are spent before determining conditions for obtaining sufficiently good crystals for conducting a three-dimensional structure analysis. It is a bottleneck in 3D structure analysis.

  On the other hand, various protein crystallization apparatuses, crystallization methods, and crystallization screening methods have been proposed in order to perform screening of protein crystallization conditions or to perform crystallization quickly and in a smaller amount of sample. .

  Some of the present inventors have also conducted a crystallization experiment on a microarray-type chip for the purpose of quickly and economically screening protein crystallization conditions with a small amount of sample. (For example, refer to Patent Document 1 and Patent Document 2).

  In the inventions described in Patent Document 1 and Patent Document 2, screening for protein crystallization conditions is performed by using a microarray. In the microarray, a gel-like material is held in each section formed by the through-hole, and each gel-like material contains a plurality of types and concentrations of protein crystallizing agents. By contacting such a microarray with a protein-containing sample, a plurality of crystallization conditions can be screened at one time. Furthermore, it can be carried out with a very small amount of protein-containing sample, which is very efficient. Examples of means for bringing the microarray into contact with the protein-containing sample include a dedicated auxiliary jig having a through-hole adapted to a desired capacity, a general dispensing instrument such as a pipetter, an automatic dispensing device, etc. The more the number of compartments held in the container, the more advantageous is the automatic dispensing device in terms of simplicity and speed.

WO03 / 053998 pamphlet JP 2005-58889 A

  The automatic dispensing devices sold by various manufacturers have various mechanisms for adjusting the dispensing amount, but generally the dispensing amount varies depending on the viscosity of the liquid used. Therefore, the automatic dispensing device itself does not have a calibration curve, and the user currently sets a calibration curve according to the liquid used. Protein-containing samples used for protein crystallization have a wide range of types and concentrations, and thus a wide range of viscosities. In applications that require accurate dispensing of protein-containing samples, such as protein crystallization, each time a protein-containing sample is changed, it is necessary to weigh the dispensed amount and adjust the automatic dispensing device.

  At this time, the dispensing amount is generally measured by measuring the mass of the protein-containing sample using a balance or the like. However, in the crystallization of the protein, the dispensing amount of one drop is as small as 0.5 μl or less. Therefore, in order to perform high-precision weighing with an electronic balance with a minimum display of 0.1 mg that is generally popular, the total mass dispensed about 10 to 100 drops is measured, and 1 from the total mass obtained is measured. It is necessary to calculate the amount of drops dispensed. As described above, the conventional method of weighing the dispensing amount is extremely complicated. The protein-containing sample may be an expensive and valuable biological sample. In such a case, it is preferable to avoid consuming a large amount of the protein-containing sample for the adjustment of the automatic dispensing device.

  Under such circumstances, there has been a demand for a weighing means that can easily weigh a dispensed amount of a protein-containing sample.

As a result of intensive investigations, the present inventors have used a weighing means having two or more weighing recesses having different volumes in order to weigh the dispensing amount of a protein-containing sample, thereby simplifying the dispensing amount. The present invention has been completed by finding out that it can be weighed.
That is, the present invention is as follows.

(1) A weighing means for weighing a dispensing amount of a protein-containing sample, the weighing means having two or more weighing recesses having different volumes.
(2) The weighing means according to (1) above, wherein the weighing recesses are arranged in order of volume.
(3) The weighing means according to (1) or (2), wherein the volume of each weighing recess is in the range of 0.001 μl to 2 μl.
(4) The weighing means according to any one of (1) to (3), wherein the weighing recess has a hemispherical or cylindrical shape.
(5) A protein crystallization apparatus comprising the weighing means according to any one of (1) to (4) above and a protein crystallization microarray having two or more recesses.
(6) The above (5), comprising: a first plate on which a protein crystallization microarray having two or more recesses is disposed; and a second plate having a recess corresponding to the recesses of the protein crystallization microarray. The device described.
(7) The apparatus according to (6) above, wherein the weighing means is provided on the first plate and / or the second plate.
(8) The protein-containing sample is dispensed into each of the concave portions of the means according to any one of (1) to (4), and the volume of the concave portion for weighing in which the dispensed sample exhibits a ground state. A method for weighing a dispensing amount of a protein-containing sample, which is a dispensing amount of the protein-containing sample.
(9) The dispensing amount of the dispensing device is adjusted based on the dispensing amount weighed by the weighing method described in (8) above, and the protein-containing sample is prepared using the dispensing device with the adjusted dispensing amount. A method for dispensing a protein-containing sample to be dispensed.

  Thus, according to the present invention, a weighing means for dispensing the protein-containing sample is provided. According to a preferred embodiment of the present invention, the amount of the protein-containing sample can be easily weighed.

Hereinafter, embodiments of the present invention will be described with reference to the drawings, but the present invention is not limited thereto.
The present invention provides a weighing means for weighing a dispensing amount of a protein-containing sample, the weighing means having two or more weighing recesses having different volumes. The present invention also provides a protein crystallization apparatus comprising such weighing means and a protein crystallization microarray having two or more recesses. Furthermore, the present invention is to dispense an equal amount of a protein-containing sample into each of two or more weighing recesses having different volumes, and to determine the volume of the weighing recess in which the dispensed sample is in a ground state. A method for weighing a protein-containing sample to be dispensed is provided.

[Protein crystallization equipment]
FIG. 1 shows an example of an apparatus for protein crystallization according to the present invention. 1 includes a first plate 11 (hereinafter sometimes referred to as a lower plate), a second plate 16 (hereinafter sometimes referred to as an upper plate), and two or more plates. The protein crystallizing microarray 14 having a recess, and the first plate and the second plate are provided with weighing means 12 and 17 for dispensing a protein-containing sample, respectively.

  In this protein crystallization apparatus, the protein crystallization microarray 14 is fitted into the protein crystallization microarray holding recess 13 provided in the lower plate 11, and the protein crystallization microarray 14 is disposed on the lower plate 11. The protein crystallizing microarray 14 holds a protein crystallizing agent-retaining gel-like substance in the recess 15. Further, the upper plate 16 has two or more recesses 18 corresponding to the recesses 15 of the protein crystallization microarray 14. A protein-containing sample is held in the recess 18.

  Further, in this protein crystallization apparatus, the protein-containing sample is dispensed into the protein-containing sample holding recess 18 provided on the upper plate 16, the lower plate 11 and the upper plate 16 are overlapped, and the lower plate 11 and the upper plate are overlapped. 16 can be fastened with fastening parts 19 through corresponding through holes 20 and 21 respectively provided at four locations. When the lower plate 11 and the upper plate 16 are overlapped, the protein-containing sample dispensed into the protein-containing sample holding recess 18 comes into contact with the protein crystallization agent holding gel-like material of the protein crystallization microarray 14, and the fastening part Fastening at 19 maintains the crystallization section.

Here, in the present invention, the “protein-containing sample” is a sample containing a target protein to be crystallized or a target protein to be specified for crystallization conditions. Examples of proteins include natural or synthetic peptides, polypeptides, proteins, and protein complexes. These substances are extracted and isolated from natural or synthetic materials, or produced by genetic engineering techniques or chemical synthesis techniques, and then combined with conventional purification methods such as solvent extraction, column chromatography, liquid chromatography, etc. It is preferable to purify the protein to be screened by using it.
“Weighing” refers to measuring the dispensing amount (volume) of a protein-containing sample dispensed by an automatic dispensing device or the like.

Hereinafter, the weighing means in a preferred embodiment of the present invention will be described in more detail.
When an automatic dispensing device is used for dispensing a protein-containing sample, it is necessary to adjust the dispensing amount of the automatic dispensing device before dispensing. As described above, conventionally, adjustment of the dispensing amount of the automatic dispensing apparatus has involved complicated weighing work and loss of the protein-containing sample.
Therefore, in the apparatus for protein crystallization shown in FIG. 1, as described above, the weighing means 12 and 17 are provided on the surfaces of the lower plate 11 and the upper plate 16 to successfully solve the conventional problems. The weighing means 12 and 17 are used when adjusting the dispensing amount of an automatic dispensing device or the like.

  FIG. 2 shows a use state of the weighing means of FIG. That is, FIG. 2 is a cross-sectional view of the weighing means 12 and shows a state in which a certain amount of protein-containing sample is dispensed into each of the five weighing recesses 12a to 12e arranged in a line in the order of volume. In FIG. 2, the volume of the weighing recess 12a is the smallest, and the volume of the weighing recess 12e is the largest. In this way, a predetermined amount of the protein-containing sample 22 is dispensed into each of the weighing recesses 12a to 12e of the weighing means 12 arranged on the lower plate 11 using an automatic dispensing device or the like, and the protein-containing sample from the side. By visually observing the level of the liquid level of 22, it is possible to visually weigh the dispensing amount of the automatic dispensing device. For example, consider a case where an equal amount of a sample whose amount is known in advance is added to each of the recesses 12a to 12e in FIG. Then, when the amount of the sample is larger than the volume of the recess, a part of the sample protrudes from the recess (12a, 12b), whereas when the amount of the sample is equal to the volume of the recess or smaller than the recess, The sample will be entirely contained in the recess (12c, 12d, 12e). Therefore, when the liquid level of the protein-containing sample 22 is viewed from the side, the liquid level is above the opening of the weighing recess in the weighing recesses 12a and 12b, and the liquid level is in the weighing recess 12c. A ground state is exhibited with respect to the opening of the weighing recess, and in the weighing recesses 12d and 12e, the liquid level is below the opening of the weighing recess. Therefore, the volume of the weighing recess 12c in which the dispensed sample is in a ground state can be regarded as the dispensing amount of the automatic dispensing device at this time.

  In a preferred embodiment of the present invention, the dispensing amount of the protein-containing sample can be easily weighed, and therefore the adjustment work of the automatic dispensing apparatus can be performed relatively easily. Moreover, in the preferable aspect of this invention, the protein containing sample consumed for adjustment of an automatic dispensing apparatus can be saved. In this case, protein crystallization or screening for crystallization conditions can be performed quickly and economically with high reliability. Here, “crystallization” refers to obtaining a protein crystal by growing or precipitating a crystal from a protein-containing sample. The “protein crystallization conditions” include, for example, the type and concentration of the protein crystallization agent, the pH of the gel after solidifying the protein crystallization agent, the composition and the degree of crosslinking, the temperature of the crystallization compartment, Crystallization time and cooling profile.

  In FIG. 2, the protein-containing sample is dispensed into all of the five weighing recesses 12a to 12e of the weighing means 12 provided on the lower plate 11, and the weighing is performed. However, the present invention is limited to this. Alternatively, the protein-containing sample may be dispensed into a part of the weighing recesses 12a to 12e to perform weighing. Further, the dispensing amount of the protein-containing sample may be weighed using all or a part of the weighing concave portion of the weighing means 17 provided on the upper plate 16, or the lower plate 11 and the upper plate 16 may be weighed. The protein-containing sample may be weighed using all or a part of the weighing recesses of both the weighing means 12 and 17 provided in.

  Further, in the protein crystallization apparatus of FIG. 1, the dispensing amount weighing means is provided in both the lower plate 11 and the upper plate 16, but the present invention is not limited to this, and the lower plate 11 or the upper plate 16. It is also possible to omit only one of the weighing means 12 or 17 by providing only one of them. When the weighing means is provided in the protein crystallization apparatus, the place where the weighing means is provided is not particularly limited, but for convenience, as shown in FIG. 1, two or more different volumes are provided near the outer periphery of the lower plate 11 and the upper plate 16. Most preferably, the recesses are arranged in one row. Further, a plurality of rows of recesses having two or more different volumes may be provided in one lower plate 11 or upper plate 16.

  Further, the weighing means 12 and 17 in FIG. 1 have 10 weighing recesses with different volumes, 10 in total. However, the number of weighing recesses of the weighing means of the present invention is not limited to this, it may be two or more, and particularly preferably 3 to 10. Moreover, according to the preferable aspect of this invention, the recessed part for weighing is arranged in order of a volume. That is, the volume of the weighing recess is gradually changed. Furthermore, according to the preferable aspect of this invention, the volume of each recessed part exists in the range of 0.001 microliters-2 microliters, More preferably, it is 0.01 microliters-1 microliter. The greater the number of weighing recesses of the weighing means, the greater the total volume change and the weighing range is expanded. In addition, there is an advantage in terms of weighing capacity, such as the weighing accuracy is improved by reducing the volume change between the weighing recesses. On the other hand, the smaller the number of weighing recesses, the lower the manufacturing cost of the weighing means and the loss of the protein-containing sample. The number of weighing recesses may be appropriately increased or decreased in consideration of user convenience.

  Further, in the weighing means 12 of FIG. 2, the shape of the weighing recess is hemispherical, but in the present invention, the shape of the weighing recess is not limited to this. For example, as shown in FIG. There may be. Further, as shown in FIG. 3B, the opening side may be formed in a columnar shape, and the bottom side may be formed in a hemispherical shape. Since the dispensed protein-containing sample is generally circular, the shape of the opening of the weighing recess is preferably circular. The shape of the weighing recess is preferably hemispherical or cylindrical, and most preferably hemispherical. Taking a hemisphere as an example, the radius is 0.078 mm, the volume is 0.001 μl, the radius is 0.985 mm, and the volume is 2 μl. Taking a cylinder as an example, the radius is 0.08 mm × depth 0.05 mm, the volume is 0.001 μl, the radius 1.03 mm × depth 0.6 mm, and the volume is 2 μl. Note that the volume of the weighing recess can be changed by appropriately changing the width (for example, diameter) and / or depth of the recess.

  Further, examples of the processing method of the weighing means 12 and 17 include removal processing represented by mechanical cutting and laser cutting, and molding processing represented by blow molding, vacuum molding, injection molding, and optical molding. Injection molding is the most suitable because the shape can be mass-produced efficiently with high reproducibility. The removal processing represented by mechanical cutting and laser cutting may cause differences between weighing means because processing errors are unavoidable, but it does not require a molding die and the initial investment can be reduced. There are advantages. Further, the transfer processing technique represented by hot embossing is suitable for molding a finer shape than other methods, and is more suitable as the amount of the target protein-containing sample decreases.

  In addition, when the material used is hydrophobic, the surface of the weighing means including the surface of the concave portion is further reduced in order to reduce the rise from the edge of the concave portion due to the surface tension of the protein-containing sample. A hydrophilization treatment may be appropriately applied to the entire surface.

Next, components other than the weighing means of the apparatus shown in FIG. 1 will be described in more detail.
In the protein crystallization apparatus of the present invention, it is preferable that the change in crystallization behavior over time can be observed through the lower plate 11 or the upper plate 16. For this reason, it is preferable that the lower plate 11 and / or the upper plate 16 include a transmission observation part with high transparency. Examples of the material of the transmission observation portion of the lower plate 11 and / or the upper plate 16 include acrylic, ABS (acrylonitrile / butadiene / styrene), polycarbonate, various transparent resins typified by polyester, and glass. As other part materials, in addition to the above-mentioned various transparent resins, various resins represented by nylon, polyamide, polyacetal, Teflon (registered trademark), various metals and alloys represented by iron, aluminum, copper, titanium and magnesium. In addition, powder metallurgy sintered bodies represented by tungsten carbide, composite resins obtained by mixing glass fibers and carbon fibers with the resin, inorganic materials represented by glass and ceramics, and the like are included, but are not limited thereto. It is not a thing. Most suitable are various transparent resins represented by acrylic, ABS, polycarbonate, and polyester, which can mold the entire apparatus including the transmission observation part of the apparatus with a single material.

  In FIG. 1, a protein crystallization microarray 14 is used, for example, for protein crystallization or screening for crystallization conditions. In the apparatus of FIG. 1, the shape of the protein crystallization microarray 14 is rectangular, but the present invention is not limited to this, and may be circular or square, for example. The thickness of the protein crystallization microarray 14 can be arbitrarily selected in consideration of the efficiency of crystallization, the ease of observation of the crystallization behavior, etc., but preferably 0.1 mm to 5 mm. Preferably, it is 0.2 mm to 2 mm.

Further, the number of the concave portions of the protein crystallizing microarray 14 is 10 or more per 1 cm ^{2 of} microarray, and preferably 10 to 200 per 1 cm ² .

  Furthermore, the material of the protein crystallization microarray 14 is preferably highly transparent so that it can be observed through transmission. Examples of such a material include glass and resin described above as materials for the upper plate and the lower plate.

  Suitable examples of the protein crystallization microarray used in the protein crystallization apparatus of the present invention include two or more tubular bodies described in WO03 / 053998 pamphlet and JP-A-2005-58889. This is a microarray. This microarray can be manufactured by regularly arranging hollow fibers and bonding them with a resin adhesive or the like. Examples of the material of the hollow fiber include a homopolymer of a methacrylate monomer and an acrylate monomer, or a copolymer thereof. Examples of the resin adhesive include a polyurethane resin adhesive. When the microarray is formed by arranging two or more tubular bodies, the concave portion of the microarray is usually a through hole penetrating the microarray.

  The protein crystallizer-retaining gel-like product is a gel-like product that retains the protein crystallization agent. The protein crystallizing agent means a compound having a function of lowering the solubility of the protein, and examples thereof include a precipitant, a pH buffering agent, and other adducts. Further, the gel-like product is not particularly limited. For example, acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, N-vinyl. One or more monomers such as pyrrolidone, hydroxyethyl methacrylate, (meth) acrylic acid, and allyl dextrin, and a polyfunctional monomer of methylenebis (meth) acrylamide or polyethylene glycol di (meth) acrylate Mention may be made, for example, of gels copolymerized in an aqueous medium. Examples of other gel-like materials include gels such as agarose, alginic acid, dextran, polyvinyl alcohol, and polyethylene glycol, and gels obtained by crosslinking these.

  The gel-like material can retain a plurality of types and concentrations of protein crystallizing agents. Here, “retaining” means immobilizing the protein crystallizing agent on the microarray. The method for retaining the protein crystallizing agent in the gel-like material is not particularly limited. For example, there is a method in which a protein crystallization agent and a polymerizable monomer that becomes a gel are mixed, and then a polymer gel is formed through a polymerization process to immobilize the protein crystallization agent on the gel.

  Furthermore, the fastening part 19 may be any part that can fasten the lower plate 11 and the upper plate 16, and the material and shape thereof are not limited. Further, the shape and number of the through holes 20 and 21 may be appropriately selected according to the material and shape of the fastening part, and are not limited. Further, when the shape of the fastening component is, for example, a clip shape, the lower plate 11 and the upper plate 16 can be fastened without providing a through hole.

  The protein crystallization apparatus according to a preferred embodiment of the present invention can be used, for example, in screening and screening of protein crystallization conditions and production of crystals for protein structure analysis in research and development in the pharmaceutical field and the like.

[Method for dispensing protein-containing samples]
In a preferred embodiment of the present invention, based on the dispensing amount weighed using the weighing means described above, the dispensing amount of the automatic dispensing device is adjusted, and the dispensing device with the adjusted dispensing amount is used to contain the protein. Dispense the sample.
The automatic dispensing device is a device that automatically dispenses a liquid protein-containing sample. The number of spouts of the automatic dispensing device is not particularly limited, but it is preferable to use an automatic dispensing device having two or more spouts. The automatic dispensing device is not particularly limited as long as an appropriate amount of protein-containing sample can be poured out, and examples thereof include one having a micropipette.

  And adjustment of the dispensing amount of an automatic dispensing apparatus is performed based on the weighed dispensing amount. Adjusting the dispensing amount of the automatic dispensing device refers to adjusting the setting of the automatic dispensing device so that the protein-containing sample can be dispensed with a preset dispensing amount.

  The dispensing amount can be adjusted, for example, by adjusting the amount of pressurization with air.

  Further, when the protein-containing sample is dispensed into the apparatus of FIG. 1 after adjustment of the automatic dispensing apparatus, the protein-containing sample is dispensed into the protein-containing sample holding recess 18. However, the present invention is not limited to this, and it may be directly dispensed into the concave portion 15 of the microarray 14 holding the protein crystallizer-holding gel-like material, or the protein-containing sample holding concave portion 18 and the microarray 14. Alternatively, it may be dispensed by appropriately distributing to both of the concave portions 15.

[Screening of protein crystallization conditions and protein crystallization]
When screening for protein crystallization conditions using the apparatus shown in FIG. 1, for example, the screening is performed as follows. First, a protein crystallization agent and a protein-containing sample are reacted in a plurality of crystallization conditions in a protein crystallization microarray. Next, the apparatus for protein crystallization is left in a sealed state or in the atmosphere under a certain temperature condition for a time sufficient for the protein to precipitate. Then, after a sufficient time has elapsed for the protein to precipitate, the state of protein crystallization is observed, and the optimum crystallization conditions for the target protein are determined.

  Furthermore, protein crystallization can be performed using a known crystallization method such as a vapor diffusion method or a hanging drop method based on the crystallization conditions determined by screening. Protein crystallization may be performed using the apparatus shown in FIG. In that case, for example, the crystallization may be continuously performed in the crystallization section where the crystals are deposited by screening. Furthermore, a crystal precipitated by screening can be collected and used as a seed crystal to produce a protein crystal by a known crystallization method. Known crystallization methods include the hanging drop method, sitting drop method, dialysis method, and batch method.

  The obtained protein crystal is recovered, and the recovered crystal is analyzed by a known method using a three-dimensional structure analysis method such as X-ray structure analysis, whereby the three-dimensional structure of the control protein can be determined.

[Example 1]
(Production of lower plate)
Using a transparent acrylic resin VA001 (manufactured by Mitsubishi Rayon Co., Ltd.) as a material, a lower plate was manufactured by injection molding. The outer dimension of the lower plate was 65 mm long × 55 mm wide × 12 mm thick, and a recess for holding a microarray for protein crystallization of 30 mm long × 20 mm wide × 0.7 mm deep was provided in the center of a 65 mm × 55 mm surface. Further, five hemispherical protein-containing sample weighing recesses were provided at intervals of 2 mm at a location of 2.5 mm parallel to the 55 mm side of the same surface. The radius of each recessed part was SR = 0.135mm, 0.17mm, 0.195mm, 0.21mm, 0.23mm in order. The volume of each recess is 0.005 μl, 0.01 μl, 0.015 μl, 0.02 μl, and 0.025 μl in order. Furthermore, four through holes (M5 female screw holes) for fastening with the upper plate were provided on the same surface.

(Production of upper plate)
A transparent acrylic resin VA001 (manufactured by Mitsubishi Rayon Co., Ltd.) was used as a material, and an upper plate was manufactured by injection molding. The outer dimensions of the upper plate are 65 mm long x 55 mm wide x 12 mm thick, and a columnar protein-containing sample holding recess of 0.7 mm diameter x 0.15 mm deep at the center of a 65 mm x 55 mm surface x 12 x vertical A total of 96 horizontal 8 pieces were provided at intervals of 2 mm in both vertical and horizontal directions. Further, five hemispherical protein-containing sample weighing recesses were provided at intervals of 2 mm at a location of 2.5 mm parallel to the 55 mm side of the same surface. The radius of each recessed part was SR = 0.265mm, 0.29mm, 0.305mm, 0.32mm, and 0.335mm in order. The volume of each recess is 0.04 μl, 0.05 μl, 0.06 μl, 0.07 μl, and 0.08 μl in order. Further, four through holes with a diameter of 5.5 mm for fastening with the lower plate were provided on the same surface.

(Weighing amount 1 of automatic dispensing device; target value 0.015 μl)
As an automatic dispensing device, a non-contact jet dispenser MJET-1 (all manufactured by Musashi Engineering Co., Ltd.) mounted on a desktop three-axis simultaneous control robot SHOTminiSL is used as a protein-containing sample, and egg white-derived lysozyme 120-02674 (Jun Wako) Dispensing of an automatic dispensing apparatus using a lysozyme aqueous solution in which 30 mg of Yakuhin Kogyo Co., Ltd. dissolved in 500 μl of ultrapure water was used as a weighing means for a protein-containing sample on the lower plate produced as described above. A weighing of the quantity was carried out.

First, the protein-containing sample is loaded into the liquid agent reservoir of the automatic dispensing device, then the air pressure reducing valve of the automatic dispensing device is set to 0.02 MPa, the timer is set to 0.004 sec, and the micrometer reading is set to 0.1 mm, and the lower plate is set. Were dispensed into five recesses for weighing protein-containing samples.
When the five protein-containing sample weighing recesses are viewed from the side of the 55 mm side of the lower plate, the protein-containing sample overflows from the SR = 0.135 mm and 0.17 mm recesses, and the SR = 0.195 mm recess shows a ground state. Presented, the SR = 0.21mm and 0.23mm concave portions were in a state where the liquid level was depressed.
Therefore, it was found that the dispensing amount of the automatic dispensing device was 0.015 μl under this condition.

(Weighing amount 2 of the automatic dispensing device; target value 0.06 μl)
Using the same automatic dispensing device and protein-containing sample as described above, and weighing the protein-containing sample on the upper plate manufactured as described above, weigh the dispensing amount of the automatic dispensing device. did.

As before, the protein-containing sample is first loaded into the liquid reservoir of the automatic dispensing device, then the air pressure reducing valve of the automatic dispensing device is set to 0.02 MPa, the timer is set to 0.006 sec, and the micrometer reading is set to 0.1 mm. The upper plate was dispensed into five concave portions for weighing protein-containing samples.
When the five protein-containing sample weighing recesses are viewed from the side of the 55 mm side of the upper plate, the protein-containing sample overflows from the SR = 0.265 mm and 0.29 mm recesses, and the SR = 0.305 mm recess is in a ground state. Presented, the liquid level was depressed in the concave portions of SR = 0.32 mm and 0.335 mm.
Therefore, it was found that the dispensing amount of the automatic dispensing device was 0.06 μl under this condition.

(Assembly of protein crystallization equipment)
Lower crystal, upper plate, automatic dispensing device, protein-containing sample, and microarray for protein crystallization as described above, protein crystallization agent with outer dimensions of 30 mm x 20 mm x 0.75 mm and diameter of 0.3 mm A protein crystallization apparatus was assembled using a structure in which a total of 96 pieces of holding gel-like materials were held in a length of 12 x 8 in width and width of 2 mm.

  First, automatic dispensing with the protein crystallization microarray fitted in the protein crystallization microarray holding recess in the center of the lower plate, the air pressure reducing valve set to 0.02 MPa, the timer set to 0.004 sec, and the micrometer reading set to 0.1 mm With the apparatus, the protein-containing sample was dispensed into all 96 protein crystallizing agent-retaining gels. This operation is intended to compensate for the case where the protein crystallizer-retaining gel-like material is irregularly thinned by about 0.01 μl.

Next, the air pressure reducing valve of the automatic dispensing device is set to 0.02 MPa, the timer is set to 0.006 sec, the micrometer reading is set to 0.1 mm, and the protein is contained in all 96 protein-containing sample holding recesses provided on the upper plate. The sample was dispensed, the lower plate and the upper plate were overlapped and fastened with four M5 bolts.
When the assembled protein crystallization apparatus was observed through the upper plate, it was confirmed that all 96 protein-containing sample holding recesses were filled with the protein-containing sample. The filling state was good without excessive or insufficient leakage of the protein-containing sample.

[Comparative Example 1]
(Weighing amount 2 of the automatic dispensing device; target value 0.06 μl)
The same automatic dispensing apparatus as in Example 1 as the protein-containing sample, and using an electronic balance A200S (manufactured by Sartorius) instead of the upper plate as the weighing means for the protein-containing sample, Weighing was performed.
As in Example 1, first, the protein-containing sample was loaded into the liquid storage part of the automatic dispensing device, then the air pressure reducing valve of the automatic dispensing device was set to 0.02 MPa, the timer was set to 0.006 sec, and the micrometer reading was set to 0.1 mm. Set and dispense 5 drops onto glass slide. When the mass of these 5 drops was measured with an electronic balance, the displayed value fluctuated between 0 to 0.2 mg and could not be measured.

[Comparative Example 2]
(Weighing amount 2 of the automatic dispensing device; target value 0.06 μl)
The automatic dispensing device and the protein-containing sample were the same as in Example 1, and the protein-containing sample was weighed in the same manner as in Comparative Example 1, and the automatic dispensing device was weighed.
100 drops were dispensed onto the slide glass under the same loading operation and conditions as in Example 1 and Comparative Example 1. When the mass of 100 drops was measured with an electronic balance, the displayed value was 5.9 to 6.2 mg.
When weighed in this way, the dispensing amount of the automatic dispensing device is determined as 0.059 to 0.062 mg, and it can be seen that the variation is large. In addition, many protein-containing samples are consumed for weighing.

[Comparative Example 3]
(Weighing amount 1 of automatic dispensing device; target value 0.015 μl)
The automatic dispensing apparatus and the protein-containing sample were the same as those in Example 1, and the protein-containing sample was weighed as in Comparative Examples 1 and 2, and the automatic dispensing apparatus was weighed.
The same loading operation as in Example 1 and Comparative Examples 1 and 2 was performed, then the air pressure reducing valve of the automatic dispensing device was set to 0.02 MPa, the timer was set to 0.004 sec, the micrometer reading was set to 0.1 mm, and the slide glass was set. One hundred drops were dispensed. When the mass of 100 drops was measured with an electronic balance, the displayed value was 1.1 to 1.6 mg.
When weighed in this way, the dispensing amount of the automatic dispensing device is determined as 0.011 to 0.016 mg, and it can be seen that the variation is large. In addition, many protein-containing samples are consumed for weighing.

It is the schematic which shows an example of the apparatus for protein crystallization of this invention. It is the schematic which shows the use condition of the measurement means of the amount of dispensing of a protein containing sample. It is the schematic which shows the example of the recessed part for weighing of a weighing means.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 ... Lower plate 12 ... Weighing means 12a-12g ... Recessed part for weighing 13 ... Recessed part for microarray holding for protein crystallization 14 ... Microarray for protein crystallization 15 ... Recessed part of microarray 16 ... Upper plate 17 ... Weighing means 18 ... Sample containing protein Retaining recess 19 ... Fastening component 20 ... Fastening through hole 21 ... Fastening through hole 22 ... Protein-containing sample

Claims (9)

  A weighing means for weighing a dispensing amount of a protein-containing sample, the weighing means having two or more weighing recesses having different volumes.   2. The weighing means according to claim 1, wherein the weighing recesses are arranged in order of volume.   The weighing means according to claim 1 or 2, wherein the volume of each weighing recess is in the range of 0.001 µl to 2 µl.   The weighing means according to any one of claims 1 to 3, wherein the weighing recess has a hemispherical or cylindrical shape.   A protein crystallization apparatus comprising the weighing means according to claim 1 and a protein crystallization microarray having two or more recesses.   The apparatus of Claim 5 provided with the 1st plate which arrange | positions the protein crystallization microarray which has a 2 or more recessed part, and the 2nd plate which has a recessed part corresponding to the recessed part of the said protein crystallization microarray.   The apparatus according to claim 6, wherein the weighing means is provided on the first plate and / or the second plate.   A protein-containing sample is dispensed into each of the recesses of the means according to any one of claims 1 to 4, and the volume of the weighing recess in which the dispensed sample is in a ground state is determined by dividing the volume of the protein-containing sample. A method for weighing the amount of a protein-containing sample to be dispensed.   The dispensing amount of the dispensing device is adjusted based on the dispensing amount weighed by the weighing method according to claim 8, and the protein-containing sample is dispensed using the dispensing device in which the dispensing amount is adjusted. A method for dispensing a protein-containing sample.

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