CN101161871B - Protein crystal board and crystallization method - Google Patents
Protein crystal board and crystallization method Download PDFInfo
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- CN101161871B CN101161871B CN2006101400384A CN200610140038A CN101161871B CN 101161871 B CN101161871 B CN 101161871B CN 2006101400384 A CN2006101400384 A CN 2006101400384A CN 200610140038 A CN200610140038 A CN 200610140038A CN 101161871 B CN101161871 B CN 101161871B
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Abstract
The present invention relates to a protein crystallized board comprising a crystallized board main part (10) and storage tank units (20) distributed on the crystallized board main part in an array form; each storage tank unit (20) is provided with a first panel (240); a groove is formed in the middle position of the first panel; the bottom of the groove is a second panel (250); the middle position or the position near one side of the second panel recesses to form a first storage tank (220); at least one dropping tank (230) is formed in the second panel (250) of at least one side of the first storage tank (220); the first panel (240) is connected with the second panel (250) through a connecting part (260); the periphery of the crystallized board main part (10) is provided with a support part (300). The invention also relates to a protein crystallization method by using the crystallized board.
Description
Technical field
The present invention relates in general to a kind of crystallization plates that is used for biological field, particularly a kind of crystallization plates that is used for the crystallization of protein of high-throughput (turnout) and meets the first three items standard of SBS standard, and the growing method that utilizes above-mentioned crystallization plates.
Background technology
Protein is the main undertaker of vital movement, is the prerequisite of understanding protein function to the understanding of its three-dimensional structure.For example, numerous disease is because some protein at the spatial false folding, forms unusual three-dimensional structure and causes.The biomacromolecule structure is the parsing of protein structure particularly; Not only human intelligible protein function, essence and the origin of inquiring into life there are significance, and important basis are provided for pathogenesis and the discovery of drug targets and the medicinal design of rationalization etc.The nearest result of protein structure DB PDB (ProteinData Bank) shows that on March 14th, 2006, existing 35579 kinds of protein structures were resolved, and wherein the protein structure more than 86% is accomplished through X-ray crystallography.It is thus clear that X-ray crystallography remains the most important means of resolving protein three-dimensional structure so far.X ray single crystal diffraction method is measured protein structure cardinal principle flow process can be divided into these five steps of clone, expression, purifying, crystallization and crystallographic structural analysis in order.Because required step is more, time loss corresponding length, plant and instrument demand are also complicated, the consumption of resolving each albumin crystal structure is still very big.According to America NI H statistical information, with regard to present level, on average resolving an albumin crystal structure needs the expense more than 100,000 dollars.The protein single crystal samples that obtains having diffracting power is the prerequisite of crystallographic structural analysis, also is " bottleneck " step that present x-ray crystal structure is resolved.Theoretically, as long as find suitable crystal growth condition, the protein of solubility can obtain high quality, be applicable to the monocrystalline of crystallographic structural analysis.But screening of albumin crystal growth conditions at present and optimization are to a great extent more as an art, and the searching of this " conditions suitable " needs a large amount of trials, carries out loaded down with trivial details primary dcreening operation and optimization.
Method of protein crystallization commonly used at present has hanging drop gas phase diffusion method, a seat gas phase diffusion method, oil to cover method etc.Hanging drop gas phase diffusion method is meant protein soln that on the sheet glass that scribbles sealing material (like Vaseline), plastic sheet or special adhesive tape dropping is to be crystallized and crystallization screening solution etc., and tips upside down on the pond liquid that contains above-mentioned crystallization screening solution.Sitting a gas phase diffusion method need drip the mixed solution of protein soln to be crystallized and crystallization screening solution in a dropping bath, and with special plastic film or rubber belt sealing.
Fig. 1 and Fig. 2 have described sessile drop method respectively and have sat the synoptic diagram that drips the method crystallization of protein, and Fig. 3 shows the stereographic map that prior art is used for 24 orifice plates of sessile drop method.
Fig. 1 is the synoptic diagram of sessile drop method.The solution that is used for the crystallization of protein screening in the big storage tank 2 comprises buffer reagent or precipitation agent etc. usually.Also comprise identical crystallization of protein screening solution and the protein soln to be crystallized that add by a certain percentage in the small droplets 4 of protein soln; Make the concentration in these solution be lower than the solution in the big storage tank 2 such as buffer reagent or precipitation agent; After sealing, make in each unit the internal medium that solution and small droplets 4 vapor diffusion between the two through big storage tank 2 reaches the running balance of carrying out crystallization of protein.
Fig. 2 is for sitting the synoptic diagram of the method for dripping.In this method, protein small droplets 4 is placed on the support of storage tank solution 2 tops, this and sessile drop method are opposite.Along with people's is to the intensification of albumen crystallisation process understanding and to a large amount of trials and the combination of all ingredients; The crystal screening conditions are increasing; The screening conditions commonly used with Hampton company are example, Screen KitI, II and Index, totally 192 screening conditions; Being commonly used to optimize crystalline additive test kit also has nearly 100 conditions, and the crystal screening conditions commonly used that other companies or laboratory can provide also have hundreds of to arrive thousands of kinds.Can carry out the screening of thousands of condition to each albumen, be prerequisite but " mass-election " of a large amount of conditions be consumption with plenty of time and reagent, and also need more albumen, a large amount of proteinic production expression and purifying are unusual difficulties under a lot of situation.
The common employing amount of carrying out protein crystal growth at present is between 0.1～4.0 μ L, and mostly the crystallization plates that screens is plastics.For high-throughput, fully automated crystal growth; Usually adopt 96 holes, 384 holes even the 1536 hole crystallization plates that meet international standard (ansi standard SBS (Society forBiomolecular Sciences)); Screening conditions are many, the application of sample amount is little, saved sample and screening reagent greatly.But because pond liquid bath and dropping bath are too little, arrange closely simultaneously, be inappropriate for very much manual operation, manually operated working efficiency has on the contrary slowed down.
And for most of laboratories, crystal growth is still accomplished by manual operations with screening is general, uses 24 traditional hole crystallization plates (shown in Figure 3), but this crystallization plates is not suitable for sitting the gas phase diffusion method of dripping.In addition; Existing market has many to the instrument and the self-reacting device that meet the SBS international standard; Like sample injector (like Multi-channel Pipette), robotization crystal growth recording geometry, and traditional 24 hole crystallization plates can not use these modern techniquies because meeting the SBS international standard.
On the other hand, these factors such as sample is volatile, reagent application of sample amount is few all have a strong impact on the repeatability of experimental result, and especially accuracy is poorer under manual operation, operation is slow.Protein and SOLUTION PROPERTIES are different in addition, the speed of protein crystal growth can from several hours to not waiting in several weeks even some months, therefore the closure for the environment of crystal growth system requires very high.But present many all ubiquity leakage problems in various degree of high-throughout crystallization plates that are applicable to.
To the problems referred to above; The state of the art in conjunction with development such as application of sample instrument and recording geometrys; And consider that at present domestic and international most experiments still adopts manual work or semi-automatic technique to carry out protein crystal growth; The present invention proposes a kind of crystallization of protein plate and method of protein crystallization, promptly be applicable to high-throughout fully automated crystal growth and observation screening system, satisfy the semi-automatic needs of most laboratory again.
Summary of the invention
Based on above-mentioned needs, one aspect of the present invention provides a kind of high throughput protein crystalline crystallization of protein plate that is used for, and comprises the crystallization plates main part; And the storage tank unit that on the crystallization plates main body, distributes with array format; Each said storage tank unit has first panel, forms a groove in the said first panel mid-way, and said bottom portion of groove is second panel; Recessed first storage tank that forms in the said second panel mid-way; In second panel of at least one side of said first storage tank, be formed with at least two dropping baths, said first panel is connected through connection section with second panel, and said crystallization plates main part has the support portion around it.
Preferably, the storage tank unit of said array format distribution is 24 or 48; And said at least two dropping baths are two, three, four, five or six.
Preferably; Said crystallization plates is processed by the high purity thermoplastics, like polyamide (PA), Vestolen PP 7052 (PP), PS (PS), acrylonitrile-butadiene-styrene copolymer (ABS), polycarbonate (PC), polymethylmethacrylate (PMMA), styrene-acrylonitrile resin (SAN), gather cyclenes nitrile polymer (COP) etc.
Preferably, said crystallization plates is sealed by Sealing strap or shrouding film.
Preferably, the volume of said first storage tank is 0.04～1.4ml, and the volume of said dropping bath is 0.2～7 μ l.
More preferably, said crystallization plates is processed by PS.
Preferably, wherein, the shape of the dropping bath and first storage tank independently is selected from the group that is made up of right cylinder, hemisphere, inverted Rotary-table or cone, cubes, rectangular parallelepiped.
More preferably; Wherein, Said crystallization plates is 24 (promptly 4 * 6) orifice plate or 48 (promptly 8 * 6) orifice plate; Its size conforms SBS standard, i.e. conformance with standard ANSI/SBS1-2004: coverage area size, ANSI/SBS2-2004: height dimension, ANSI/SBS3-2004 bottom outside flange dimension, and to ANSI/SBS4-2004: the hole site standard is improved.
More preferably, wherein, said crystallization plates is 48 (promptly 8 * 6) orifice plate comprises the crystallization plates main part, and the total length of first panel of said main part is 120.5～125.5mm, and total width is 78.5～83.5mm; Wherein, the unitary length of each storage tank is that 13.5～15.5mm, width are 6.5～7.5mm; Distance between first panel and second panel upper surface is 2～8mm; The width of first storage tank is that 3.0～6.0mm, length are that 6.5～7.5mm, the degree of depth are 3.5～9.5mm; The upper end diameter R of dropping bath
OnBe 3.0>=R
On>=1.5mm, lower end diameter R
DownBe 2.5>=R
Down>=0.5mm, the degree of depth of dropping bath is 0.2～1.0mm; The width of first panel around in the storage tank unit is 1.5～4.5mm, and is 5.5～10.5mm at the width of first panel of the storage tank unit periphery of the most peripheral of crystallization plates main part; The total height of said support portion is 13.8～14.6mm.
More preferably, wherein, said crystallization plates is 24 (promptly 4 * 6) orifice plate comprises the crystallization plates main part, and the total length of first panel of said main part is 120.5～125.5mm, and total width is 78.5～83.5mm; Wherein, the unitary length of each said storage tank is that 15.0～16.5mm, width are 15.0～16.5mm; Distance between said first panel and second panel upper surface is 2～8mm; The length of first storage tank is that 15.0～16.5mm, width are that 5～9mm, the degree of depth are 3.5～9.5mm; The upper end diameter R of dropping bath
OnBe 3.0>=R
On>=1.5mm, lower end diameter R
DownBe 2.5>=R
Down>=0.5mm, the degree of depth of dropping bath is 0.2～1.0mm; The width of first panel around in the storage tank unit is 1.5～3.0mm, and is 5.5～10.0mm at the width of first panel of the storage tank unit periphery of the most peripheral of crystallization plates main part; The total height of support portion is 13.8～14.6mm.More preferably; Wherein, The support and connection portion of support portion by last support portion, following support portion and about connecting, said support portion constitutes; The height of wherein going up the support portion is 6.1～12.5mm, the height of support portion is 2.0～8.0mm down, and the width of the support and connection portion of support portion is 1.0～4.0mm about connecting.Again preferably, wherein, the height of last support portion is 7.6～8.9mm, the height of support portion is 2.7～6mm down, and the width of the support and connection portion of support portion is 1.5～3.0mm about connecting.
Preferably, the end of first panel of said crystallization plates on its width has two lead angle α and β respectively; And the last support portion of said crystallization plates, and support and connection portion also have lead angle α and β respectively corresponding to first panel of crystallization plates.More preferably, lead angle α and β are 135+/-10 °.
Preferably, said crystallization plates further comprises upper body, and said housing is made up of the upper surface and a plurality of side that adapt with said crystallization plates main part, and is supported on the said support portion, is used for the dustproof of said crystallization plates and piles up.
According to another aspect of the present invention, a kind of method of utilizing the high throughput protein crystallization plates to carry out crystallization of protein is provided, has may further comprise the steps:
A. the crystallizing pond liquid by being selected from buffer reagent, precipitation agent, washing agent or combinations thereof with proper volume joins in first storage tank in each storage tank unit;
B. with solution identical among proper volume and the step a with to carry out the crystalline protein soln and join respectively according to a certain percentage at least one dropping bath in each said storage tank unit, also can in dropping bath, add additive, washing agent or its combination as required simultaneously;
C. utilize Sealing strap or shrouding film to cover on unitary first panel of said storage tank; Each the storage tank unit seal inside that will be on said crystallization plates main body distributes with array format, and in each storage tank unit the inner running balance environment of keeping crystallization of protein that forms;
D. this crystallization plates is placed under the temperature of crystallization of protein and carry out protein crystallization experiments.
According to a further aspect of the invention, provide another kind of and utilized the high-throughput crystallization plate to carry out the method for crystallization of protein, may further comprise the steps:
A. the crystallizing pond liquid that is selected from buffer reagent, precipitation agent, washing agent or combinations thereof with proper volume joins in first storage tank in each storage tank unit;
B. with proper volume with step a in identical solution with to carry out the crystalline protein soln and be added drop-wise on Sealing strap or the shrouding film according to the unitary distribution situation of the storage tank of said crystallization plates according to a certain percentage, also can in above-mentioned drop, add additive, washing agent or its combination as required simultaneously;
C. said Sealing strap or shrouding film are inverted and are covered on unitary first panel of said storage tank; The inner sealing in each storage tank unit that utilizes said Sealing strap or shrouding film to be provided on the said crystallization plates main body to distribute with array format, and in each storage tank unit the inner running balance environment of keeping crystallization of protein that forms;
D. this crystallization plates is placed under the temperature of crystallization of protein and carry out protein crystallization experiments.
Wherein, the protein soln in each storage tank unit can be identical or different; And the protein soln in the different storage tank unit also can be identical or different.
Wherein, the crystallization of protein process is monitored by robotization protein crystal growth recording geometry, photographic camera, pick up camera or manual work.
Alternatively, in the solution of step b, can further add other additive.Preferably, said at least two dropping baths are a plurality of, in second panel of the said first storage tank one or both sides, are formed with a plurality of dropping baths.
Because according to high throughput protein crystallization plates size conforms SBS of the present invention (Societyfor Biomolecular Sciences) standard; Be conformance with standard ANSI/SBS1-2004: coverage area size, ANSI/SBS2-2004: height dimension, ANSI/SBS3-2004 bottom outside flange dimension; And to ANSI/SBS4-2004: the hole site standard is improved; Thereby can be advantageously used in mechanized operation, and it is simple in structure, and easy to use.Formed the internal medium of carrying out the running balance of crystallization of protein between crystallization of protein plate according to the present invention first storage tank and at least two dropping baths in each storage tank unit, simultaneously in different storage tank unit inner each self-forming air seal.
Crystallization plates provided by the invention both had been suitable for the manual operations of crystal growth and screening in most of laboratories, was suitable for sitting the gas phase diffusion method of dripping again.In addition; The structure of crystallization plates provided by the invention and in the market instrument that meets the SBS international standard and self-reacting device; Be complementary like sample injector (like Multi-channel Pipette), robotization crystal growth recording geometry etc., thereby can utilize these modern techniquies that meet the SBS international standard.
Utilization crystallization of protein plate according to the present invention carries out crystallization of protein; Because there are a plurality of dropping baths each storage tank unit, can carry out the crystallization condition screening of several kinds of different proteins simultaneously, reduce the reagent dosage of average each protein crystallization condition screening; Thereby can reduce the cost that protein screens; And because each dropping bath or the dispersive in each storage tank unit, perhaps adjacent distance between the two is greater than the distance between each dropping bath of the prior art, for manual operations provides bigger working space; This crystal slab is observed the SBS standard simultaneously, can operate with automatic equipment.In addition, the distance between each storage tank unit is bigger, and it is poor to have overcome present many high-throughput crystallization plate stopping propertys to a great extent, the problem of leakage.
Should be appreciated that above generality is described and following detailed description all is from enumerating and illustrative purpose, is in order the present invention to be provided further explanation, to be not limited to the present invention.
Description of drawings
The accompanying drawing that constitutes a specification sheets part helps further to understand the present invention, and these accompanying drawings have illustrated some embodiments of the present invention, and can be used for explaining principle of the present invention with specification sheets.
Fig. 1 shows the synoptic diagram of crystallization of protein of the sessile drop method of prior art;
The seat that Fig. 2 shows prior art drips the synoptic diagram of the crystallization of protein of method;
Fig. 3 shows the synoptic diagram of 24 orifice plates of the sessile drop method of prior art;
Fig. 4 shows the stereographic map according to a kind of crystallization of protein plate of the present invention (48 * 2, i.e. (8 * 6) * 2 crystallization plates);
Fig. 5 shows the vertical view according to a kind of crystallization of protein plate of the present invention (48 * 2, i.e. (8 * 6) * 2 crystallization plates);
Fig. 6 shows according to the sectional side elevation of a kind of crystallization of protein plate of the present invention (48 * 2, i.e. (8 * 6) * 2 crystallization plates) along the capable dropping bath medullary ray of A;
Fig. 7 shows according to the sectional side elevation of a kind of crystallization of protein plate of the present invention (48 * 2, i.e. (8 * 6) * 2 crystallization plates) along the dropping bath medullary ray of the 1st row;
Fig. 8 shows a unitary sectional side elevation of storage tank according to a kind of crystallization of protein plate of the present invention (48 * 2, i.e. (8 * 6) * 2 crystallization plates);
Fig. 9 shows according to a kind of crystallization of protein plate of the present invention (48 * 2, i.e. (8 * 6) * 2 crystallization plates) in two unitary partial longitudinal section of continuous storage tank of crystallization plates body rim;
Figure 10 a shows the stereographic map according to another kind of crystallization of protein plate of the present invention (24 * 3, i.e. (4 * 6) * 3 crystallization plates);
Figure 10 b shows the vertical view according to another kind of crystallization of protein plate of the present invention (24 * 3, i.e. (4 * 6) * 3 crystallization plates);
Figure 11 shows the sectional side elevation according to another kind of crystallization of protein plate of the present invention (24 * 3, i.e. (4 * 6) * 3 crystallization plates) delegation's dropping bath medullary ray in capable along A;
Figure 12 shows according to the sectional side elevation of another kind of crystallization of protein plate of the present invention (24 * 3, i.e. (4 * 6) * 3 crystallization plates) along the dropping bath medullary ray of the 1st row;
Figure 13 shows the vertical view according to another crystallization of protein plate of the present invention (24 * 6, i.e. (4 * 6) * 3 crystallization plates);
Figure 14 shows the possible plot plan of dropping bath in the storage tank unit of the symmetric crystallization of protein plate of mirror image according to the present invention;
Figure 15 shows the sectional side elevation of the symmetric crystallization of protein plate of non-mirror image according to the present invention along the capable dropping bath medullary ray of A, and the corresponding possible plot plan of dropping bath in a storage tank unit;
Figure 16 shows the partial perspective view of 48 * 2 (i.e. (4 * 6) * 2) crystallization plates that is used for crystallization of protein among Fig. 4;
Figure 17 shows 48 * 2 among Figure 16, and promptly (8 * 6) * 2 crystallization plates is at the enlarged photograph that utilizes jointless Sealing strap (adhesive tape) adjacent inside, two storage tank unit sealing situation separately when sealing;
Figure 18 shows 48 * 2 among Figure 16, and promptly (8 * 6) * 2 crystallization plates is utilized in the enlarged photograph of Sealing strap (adhesive tape) adjacent inside, two the storage tank unit sealing situation separately when sealing that overlaps on first panel between two adjacent storage tank unit;
Figure 19 shows 48 * 2 among Figure 16, and promptly (8 * 6) * 2 crystallization plates is utilized in the synoptic diagram of the Sealing strap (adhesive tape) that the Sealing strap (adhesive tape) that overlaps on first panel between two adjacent storage tank unit overlaps when sealing;
Figure 20 shows crystallization plates of the prior art at the enlarged photograph that utilizes jointless Sealing strap (adhesive tape) adjacent inside, two storage tank unit sealing situation separately when sealing;
Figure 21 shows the enlarged photograph that crystallization plates of the prior art is utilized in Sealing strap (adhesive tape) adjacent inside, two the storage tank unit sealing situation separately when sealing that overlaps on first panel between two adjacent storage tank unit; And
Figure 22 shows 48 * 2 among Figure 16; Promptly (8 * 6) * 2 crystallization plates utilize jointless Sealing strap (adhesive tape) separately sealing situation of adjacent inside, two storage tank unit when sealing, a unitary Sealing strap of storage tank utilizing blade that the Sealing strap of one of them top, storage tank unit is opened and will the be opened enlarged photograph after sealing again; Wherein the figure representative in left side seals, cuts and take out crystal and the state of Sealing strap during seal operation again, and sealing, the cutting that the figure representative on right side is carried out also taken out crystal and the enlarged photograph of Sealing strap sealing situation during seal operation again.
Embodiment
Below provided various descriptions for the detailed description of some embodiment to specific embodiment of the present invention.But the present invention can be implemented the multitude of different ways that claim limited and covered.This embodiment will combine accompanying drawing to carry out, and identical in the accompanying drawings or corresponding key element or parts or position are represented with same label.
Referring to Fig. 4 to Figure 16, according to an aspect of the present invention, in a specific embodiment of the present invention; A kind of high throughput protein crystalline crystallization of protein plate that is used for is provided; Comprise crystallization plates main part 10, and the storage tank unit 20 that on the crystallization plates main body, distributes with array format, each storage tank unit 20 has first panel 240; Form a groove in the first panel mid-way; This bottom portion of groove is second panel 250, in the second panel mid-way or near recessed first storage tank 220 that forms in the position of a side, in second panel 250 of first storage tank, 220 at least one sides, is formed with at least two dropping baths 230; First panel 240 is connected through connection section 260 with second panel 250, and crystallization plates main part 10 has support portion 300 around it.Preferably, support portion 300 is by last support portion 320 and following support portion 340 and be connected up and down that the support and connection portion 360 of support portion constitutes.
Referring to Fig. 4 to Fig. 9 and Figure 14～Figure 15, in a specific embodiment (48 (8 * 6) orifice plate), at least two dropping baths 230 are a plurality of, can be 2 (Fig. 4 to Fig. 9, Figure 14), 3 (Figure 15), 4 (Figure 14), 6 (Figure 15) or any suitable number; A plurality of dropping baths 230 are recessed in second panel 250 of first storage tank, 220 both sides.
Referring to Figure 10 to Figure 15, in another specific embodiment (24 (4 * 6) orifice plate), at least two dropping baths 230 are a plurality of, can be 2 (Figure 14), 3 (Figure 16), 4 (Figure 14), 5 (Figure 15), 6 (Figure 10 or Figure 15) or any suitable a plurality of; A plurality of dropping baths 230 are recessed in second panel 250 of first storage tank, 220 1 sides.
In another specific embodiment, the storage tank unit 20 that distributes with array format in the crystallization plates main part 10 is 24, is 4 * 6 (that is, 24 orifice plates are referring to Figure 10 to 15); In another specific embodiment, the storage tank unit 20 that array format distributes is 48, is 8 * 6 (that is, 48 orifice plates are referring to Fig. 4 to 9); In another specific embodiment, the storage tank unit 20 that array format distributes is 96, be 8 * 12 (that is, and 96 orifice plates, not shown), in a storage tank unit 20, at least two dropping baths 230 are two, three, four, five or six.
In one embodiment, first storage tank 220 is shaped as right cylinder, cubes, rectangular parallelepiped, hemisphere, inverted Rotary-table or cone; Dropping bath 230 be shaped as right cylinder, cubes, rectangular parallelepiped, hemisphere, inverted Rotary-table or cone.
In one embodiment, the volume of first storage tank 220 is 0.04～1.4ml, and the volume of dropping bath 230 is 0.2～7 μ l.
Crystallization plates is processed by being not limited to following material; Promptly process, like polyamide (PA), Vestolen PP 7052 (PP), PS (PS), acrylonitrile-butadiene-styrene copolymer (ABS), polycarbonate (PC), polymethylmethacrylate (PMMA), styrene-acrylonitrile resin (SAN), gather cyclenes nitrile polymer (COP) or the like by the high purity thermoplastics.Preferred polymeric materials Vilaterm-acrylonitrile resin, Vestolen PP 7052 etc.Most preferred polymeric materials is a PS.In one embodiment, crystallization plates is sealed by shrouding film or Sealing strap, is preferably sealed by Sealing strap.In one embodiment; Crystallization plates further comprises the upper body (not shown); This housing is made up of the upper surface and a plurality of side that adapt with crystallization plates main part 10, and is supported on the support and connection portion 360 of crystallization plates, is used for the dustproof of crystallization plates and piles up.
In a specific embodiment (48 (promptly 8 * 6 * 2 is mirror symmetry) orifice plate), the crystallization of protein plate comprises: crystallization plates main part 10, and the total length of first panel 240 of main part is 120.5～125.5mm, total width is 78.5～83.5mm; The length of each storage tank unit 20 is that 13.5～15.5mm, width are 6.5～7.5mm; Wherein the length of first storage tank 220 is that 6.5～7.5mm, width are that 3.0～6.0mm, the degree of depth are 3.5～9.5mm; And the degree of depth of dropping bath 230 is 0.2～1.0mm, and diameter is 1.6～2.4mm; The total height of support portion 300 is 13.8～14.6mm; The width of first panel 240 in the storage tank unit around 20 is 1.5～4.5mm, and the width of first panel 240 around the storage tank unit 20 of the most peripheral of crystallization plates main part 10 is 5.5～10.5mm; The height of last support portion 320 is 6.1～12.5mm, the height of support portion 340 is 2.0～8.0mm down, and connects the width 1.0～4.0mm of the support and connection portion 360 of support portion up and down.
In a preferred embodiment, last support portion 320 highly is 7.6～8.9mm, support portion 340 highly is 2.7～6.0mm down, and the width of the support and connection portion 360 of support portion is 1.5～3.0mm about connecting.
More preferably; The length of each storage tank unit 20 be 14.5+/-0.25mm, width be 7.25+/-0.25mm; The length of first storage tank 220 be 7.25+/-0.5mm, width be 4.0+/-0.5mm, the degree of depth be 8.25+/-0.5mm, the degree of depth of dropping bath 230 be 0.4+/-0.1mm, for the upper and lower ends diameter be respectively 2.2+/-0.3mm and 1.7+/-0.3mm round table-like; The height of last support portion 320 be 8.25+/-0.5mm, down the height of support portion 340 be 6.10+/-0.38mm, and connect up and down the width of the support and connection portion 360 of support portion be 1.8+/-0.5mm.
Another specific embodiment (in 24 (i.e. 4 * 6 * n) orifice plates), the crystallization of protein plate comprises: crystallization plates main part 10, the total length of first panel 240 of main part is 120.5～125.5mm, total width is 78.5～83.5mm; The length of each storage tank unit 20 is that 15.0～16.5mm, width are 15.0～16.5mm, and wherein the length of first storage tank 220 is that 15.0～16.5mm, width are that 5～9mm, the degree of depth are 3.5～9.5mm, and the upper end diameter R of dropping bath (230)
OnBe 3.0>=R
On>=1.5mm, lower end diameter R
DownBe 2.5>=R
Down>=0.5mm, the degree of depth of dropping bath is 0.2～1.0mm; The total height of support portion 300 is 13.8～14.6mm; The height of last support portion 320 is 6.1～12.5mm, the height of support portion 340 is 2.0～8.0mm down, and the width of the support and connection portion 360 of support portion is 1.0～4.0mm about connecting; The width of first panel 240 in the storage tank unit around 20 is 1.5～3.0mm, and the width of first panel 240 around the storage tank unit 20 of the most peripheral of crystallization plates main part 10 is 5.5～10.0mm.
In a preferred embodiment, last support portion 320 highly is 7.6～8.9mm, support portion 340 highly is 2.7～6.0mm down, and the width of the support and connection portion 360 of support portion is 1.5～3.0mm about connecting.
More preferably; The length of each storage tank unit 20 be 16.0+/-0.25mm, width be 16.0+/-0.25mm; The length of first storage tank 220 be 16.0+/-0.5mm, width be 8.0+/-0.5mm, the degree of depth be 8.25+/-0.5mm, the degree of depth of dropping bath 230 be 0.4+/-0.1mm, for the upper and lower ends diameter be respectively 2.2+/-0.3mm and 1.7+/-the round table-like or degree of depth of 0.3mm be 1.0+/-0.2mm, diameter be 2.0+/-0.3mm closely hemispherical.The height of last support portion 320 be 8.25+/-0.5mm, down the height of support portion 340 be 6.10+/-0.38mm, and connect up and down the width of the support and connection portion 360 of support portion be 2.8mm+/-0.5mm.
In another specific embodiment, crystallization plates is sealed by the shrouding film.
Like Fig. 4, Fig. 6, Fig. 7, Fig. 9, Figure 10 a, Figure 11 and shown in Figure 12, support portion 300 is by last support portion 320, support portion 340 and connect up and down that the support and connection portion 360 of support portion 320,340 constitutes down.
Like Fig. 5, Figure 10 a, Figure 10 b and shown in Figure 13, the end of first panel 240 on its width of crystallization plates has two lead angle α and β respectively.Correspondingly, here last support portion 320, and support and connection portion 360 also have lead angle α and β respectively.Preferably, above-mentioned lead angle α and β are 135+/-10 °.More preferably, the length of first panel 240 between lead angle α, the β is 8.0～10.0mm, most preferably, the length of first panel 240 between lead angle α, the β be 9.0+/-0.5mm.
According to a further aspect in the invention, in a specific embodiment of the present invention, provide a kind of seat that utilizes the high throughput protein crystallization plates to carry out to drip the method method of protein crystallization, may further comprise the steps:
A. the crystallizing pond liquid 2 by buffer reagent, precipitation agent, washing agent etc. or combinations thereof with proper volume joins in first storage tank 220 in each storage tank unit 20;
B. solution identical among proper volume and the step a is joined respectively at least one dropping bath 230 in each storage tank unit 20 with carrying out the crystalline protein soln according to a certain percentage, also can in dropping bath, add additive, washing agent or its combination as required simultaneously;
C. utilize Sealing strap or shrouding film to cover on first panel 240 of said storage tank unit 20; Each storage tank unit 20 that will on said crystallization plates main body, distribute with array format seals, and forms the running balance environment of keeping crystallization of protein in each inside, storage tank unit;
D. this crystallization plates is placed under the temperature of crystallization of protein and carry out protein crystallization experiments.
According to a further aspect in the invention, a kind of sessile drop method method of protein crystallization that utilizes the high throughput protein crystallization plates to carry out is provided in a specific embodiment of the present invention, has may further comprise the steps:
A. the crystallizing pond liquid 2 by buffer reagent, precipitation agent, additive, washing agent etc. or combinations thereof with proper volume joins in first storage tank 220 in each storage tank unit 20;
B. with proper volume with step a in identical solution be added drop-wise on Sealing strap or the shrouding film 6 according to the distribution situation of the storage tank unit 20 of crystallization plates 10 according to a certain percentage with carrying out the crystalline protein soln, also can in above-mentioned drop, add additive, washing agent or its combination as required simultaneously;
C. said Sealing strap or shrouding film 6 are inverted and are covered on first panel 240 of storage tank unit 20; Utilize Sealing strap or shrouding film to be provided on the crystallization plates main body 10 sealing of each the storage tank unit 20 that distributes with array format, and in each storage tank unit the inner running balance environment of keeping crystallization of protein that forms;
D. this crystallization plates is placed under the temperature of crystallization of protein and carry out protein crystallization experiments.
In one embodiment, the protein soln in each storage tank unit 20 is identical, and in another specific embodiment, the protein soln in each storage tank unit 20 is different.
In one embodiment, utilize shrouding film or Sealing strap to cover on storage tank unit 20 first panel 240 all around, seal each storage tank unit 20.
In one embodiment, crystallization of protein process or result are monitored by robotization crystallization recording geometry.In another embodiment, crystallization of protein process or result are monitored by photographic camera.In another embodiment, crystallization of protein process or result are monitored by pick up camera.In another embodiment, crystallization of protein process or result are monitored by manual work.
Should be appreciated that; Though the above-mentioned crystallization plates that is used for crystallization of protein of the present invention is that example is illustrated to sit the method for dripping or sessile drop method only; But the above-mentioned crystallization plates that is used for crystallization of protein is not limited in crystallization of protein, purposes such as it also is applicable to, and other protein is cultivated, protein staining experiment, proteinic specificity combination experiment.Especially, be applicable to that all utilize in the experimental study that 24 holes, 48 holes or 96 hole crystallization plates are carried out.
Because the scantlings of the structure of crystallization plates of the present invention meets three of SBS (Society forBiomolecular Sciences) standard at least; Promptly meet ANSI/SBS1-2004, ANSI/SBS2-2004, ANSI/SBS3-2004 standard, and the ANSI/SBS4-2004 standard is improved.High throughput protein crystallization plates according to the present invention can be advantageously used in mechanized operation, and promptly the robot manipulation can carry out manual operation again.Crystallization plates according to the present invention is simple in structure; Easy to use; And formed the internal medium of carrying out the running balance of crystallization of protein between first storage tank in each storage tank unit and at least two dropping baths, simultaneously in different storage tank unit inner each self-forming air seal.
Because crystallization of protein plate according to the present invention all has one first storage tank and a plurality of dropping bath in each storage tank unit; Therefore can be simultaneously under identical condition, identical protein concn, identical buffer reagent and identical precipitation agent condition, obtain parallel experimental data; 2,3,4,5 or 6 groups of parallel laboratory test data can be obtained simultaneously, thereby the safety of parallel laboratory test can be verified.
Simultaneously; In different storage tank unit; Can carry out the experiment of some groups of different conditions simultaneously again; Like different protein concns, different buffer reagent kind and concentration, different precipitation agent kind and concentration, can carry out the experiment of conditional filtering of the crystallization of protein of some groups of (as 24 or 48 groups) different conditions simultaneously, accelerated the breakneck acceleration of protein crystallization condition.And the different dropping baths of same storage tank can drip different protein, carry out the parllel screening of many histones matter, thereby improve the efficient of screening greatly, have finally realized high-throughout protein crystallization condition screening.24 (4 * 6) orifice plates provided by the invention or 48 (8 * 6) orifice plate had both gone for manual operations, were applicable to the mechanical automation operation again.
In addition; The width of first panel 240 of crystallization of protein plate provided by the invention between two adjacent storage tank unit 10 is much larger than the width of prior art; Wherein the width of first panel 240 is at least 1.5mm, and therefore, feasible subregion sealing to crystallization plates becomes possibility.That is, equipping after filling out protein soln in each row or each is that Sealing strap capable of using (adhesive tape) carries out the maybe sealing of this row of this row, and the variation of having grown the strength of solution that has caused owing to the time when having avoided manual operation thus makes the problem of poor repeatability of crystallization experiment.Thereby of the present invention 24 or 48 orifice plates are particularly suited under the manual operation condition, to the subregion sealing of crystallization plates, have guaranteed the resistance to air loss of each 20 inside, adjacent storage tank unit simultaneously.
In addition; Like Figure 14 and shown in Figure 15; Different dropping bath 230 or be positioned at the not homonymy of first storage tank 220 in each storage tank unit 10 in the crystallization of protein plate of the present invention perhaps is positioned at the crystallization plates of 230 distances of adjacent dropping bath of first storage tank, 220 the same sides greater than prior art, therefore sits the method for dripping when carrying out crystallization of protein utilizing; Can be implemented in top dropping liquid exactly; Avoid the mistake of protein liquid to add, leaked and add or mix, and after adding crystallization screening solution and protein soln to be crystallized, can carry out reliable sample sealing, thereby improved the efficient of protein crystal.
The symmetric crystallization plates of embodiment 1 48 * 2 mirror images
Referring to Fig. 4 to Fig. 9; A kind of symmetric crystallization of protein plate of high throughput protein crystalline 48 * 2 mirror images that is used for is shown; Its size conforms SBS standard; That is: meet the standard of ANSI/SBS1-2004 coverage area size, ANSI/SBS2-2004 height dimension and ANSI/SBS3-2004 bottom outside flange dimension and to ANSI/SBS4-2004: the hole site standard is improved, and the width of first panel 240 that each storage tank unit 20 has improves on the basis of SBS standard.It is processed by PS.48 * 2 crystallization plates of present embodiment are the crystallization plates of eight row * six row * 2 (promptly 8 * 6 * 2), are different from the crystallization plates of six row * eight row * 2 (promptly 6 * 8 * 2) of prior art.
48 * 2 (promptly 8 * 6 * 2) symmetric crystallization of protein plate of mirror image of present embodiment comprises crystallization plates main part 10, the total length of first panel of this main part be 124+/-1mm, total width be 82+/-1mm;
The storage tank unit 20 that on crystallization plates main body 10, distributes, the length of each said storage tank unit 20 with 8 * 6 array formats be 14.5+/-0.25mm, width be 7.25+/-0.25mm; Each storage tank unit 20 forms a groove in first panel, 240 mid-ways; At this bottom portion of groove is second panel 250; One first storage tank 220 of recessed formation in position, second panel, 250 middle; In second panel 250 of first storage tank, 220 symmetrical both sides, be formed with two dropping baths 230; First panel 240 is connected through connection section 260 with second panel 250, and crystallization plates main part 10 has support portion 300 around it, and this support portion is by last support portion 320, support portion 340 and connect up and down that the support and connection portion 360 of support portion constitutes down.
Wherein, the distance between first panel 240 and second panel, 250 upper surfaces be 3.0+/-0.5mm; The width of first storage tank 220 be 4.0+/-0.5mm, the length of first storage tank 220 be 7.25+/-0.5mm, the degree of depth of first storage tank 220 be 8.25+/-0.5mm; The upper end diameter R of dropping bath 230
OnBe 2.2+/-0.3, lower end diameter R
DownFor 1.7+/-0.3mm, the degree of depth of dropping bath 230 be 0.4+/-0.1mm, for round table-like; Between the adjacent two storage tank unit 20 along the width of first panel 240 of crystallization plates length direction be 3.5+/-0.25mm, along the width of first panel 240 of crystallization plates width be 1.75+/-0.25mm; The total height of support portion 300 be 14.35+/-0.25mm, the height of wherein going up support portion 320 be 8.25mm+/-0.5mm, down the height of support portion 340 be 6.10+/-0.38mm and the width of support and connection portion 360 be 1.8+/-0.5mm.
The end of first panel 240 on its width of crystallization plates has two lead angle α and β for 135+/-10 ° respectively; And the last support portion 320 of crystallization plates, and support and connection portion 360 also have the lead angle α and the β of 135+/-10 ° respectively in corresponding position.
The length of first panel 240 between lead angle α, the β be 9.0+/-0.5mm.
Wherein, the width of first panel 240 between two adjacent storage tank unit 20 is about 3.5mm, its width much larger than crystallization of protein plate of the prior art (about 0.5mm).Therefore; Utilize of the present invention 48 * 2 (promptly 8 * 6 * 2) crystallization of protein plate after each row or each row storage tank unit filling protein finish, to carry out each row or the unitary sealing of each row storage tank, and real sealing fully can be realized with storage tank unit on every side in each the storage tank unit after the sealing.The photo that among the application 48 * 2 (promptly 8 * 6 * 2) mirror image symmetry crystallization of protein plate and crystallization of protein plate of the prior art each storage tank unit after sealing seal situation has been shown in Figure 17～Figure 21.
Can find out from Figure 17 and Figure 20; When of the present invention 48 * 2 (promptly 8 * 6 * 2) crystallization plates utilize jointless Sealing strap (adhesive tape) when sealing inside, adjacent two storage tank unit sealing situation separately be better than the sealing situation of the crystallization plates of prior art, but the latter has also realized adjacent inside, two storage tank unit sealing separately basically.
But can find out from Figure 18 and Figure 21; The Sealing strap (adhesive tape) that is utilized in overlap joint on first panel 240 between two adjacent storage tank unit when of the present invention 48 * 2 (promptly 8 * 6 * 2) crystallization plates is when sealing; Inside, adjacent two storage tank unit sealing situation separately is much better than the sealing situation of the crystallization plates of prior art; The former has realized the sealing fully separately of inside, adjacent two storage tank unit, but the latter leakage occurred between two adjacent storage tank unit.
Figure 19 shows the synoptic diagram that 48 * 2 among Figure 16 (promptly 8 * 6 * 2) mirror image symmetry crystallization plates is utilized in the Sealing strap (adhesive tape) that the Sealing strap (adhesive tape) that overlaps on first panel between two adjacent storage tank unit overlaps when sealing; From figure, find out,, therefore make to form sealing above that between two Sealing straps (adhesive tape) that overlap because 48 * 2 crystallization plates of the present invention have first panel 240 wideer than the crystallization plates of prior art between two adjacent storage tank unit.
(promptly 8 * 6 * 2) crystallization of protein plate is that mirror image is symmetric because 48 * 2 in the present embodiment; The position of two dropping baths 230 in each storage tank unit 20 is recessed in second panel 250 of first storage tank, 220 both sides fully symmetrically with respect to first storage tank 220; And 48 * 2 crystallization plates of the present invention have first panel 240 wideer than the crystallization plates of prior art between two adjacent storage tank unit; Therefore make and form sealing above that between the Sealing strap (adhesive tape) that overlaps; Thereby; Crystallization of protein plate of the present invention had both been realized adjacent inside, two storage tank unit positiver sealing separately, in each storage tank unit, identical crystallization condition was provided again, thereby had guaranteed crystallization result's safety and circulation ratio.
In addition; (promptly 8 * 6 * 2) crystallization of protein plate is that mirror image is symmetric because 48 * 2 in the present embodiment; Two dropping baths 230 are arranged in second panel 250 of first storage tank, 220 both sides of each storage tank unit 20, thereby have avoided in the application of sample process, wanting the mutual seepage of crystalline protein soln between two dropping baths 230.
Enlarged photograph after separately sealing situation of adjacent inside, two storage tank unit when Figure 22 shows 48 * 2 among Figure 16 (promptly 8 * 6 * 2) crystallization plates and utilizes jointless Sealing strap sealing, a unitary Sealing strap of storage tank that utilizes blade that the Sealing strap of one of them top, storage tank unit is opened and will be opened seal again; Wherein the figure representative in left side seals, cuts and take out crystal and the state of Sealing strap during seal operation again, and the figure representative on right side seals, cuts and take out crystal and the enlarged photograph of Sealing strap sealing situation during seal operation again.
As can be seen from the figure; The Sealing strap on one of them storage tank unit of covering with excellent sealing utilizes after blade cuts comes; Can take out the crystalline operation, and after accomplishing this operation, can carry out excellent sealing to this storage tank unit once more.
Wherein, show the sealing of jointless Sealing strap on adjacent storage tank unit in the left upper portion frame of broken lines.Show in the dashed middle line frame of left side and utilize the Sealing strap that seals on one of them storage tank unit of blade cuts; Sealing strap is opened, and utilization pipettes crystalline instrument (like Cryo-loop) and shifts out crystal.Show the sealing of Sealing strap on the storage tank unit of opening in the lower dotted line frame of left side.Accordingly, to show the sealing condition of jointless Sealing strap between adjacent storage tank unit good for right upper portion.Storage tank unit and the sealing condition of Sealing strap on adjacent with it storage tank unit when showing Sealing strap in the middle of the right side and opening are good.It is good that lower right side shows the again sealing condition of Sealing strap on the storage tank unit of opening.
And the crystallization plates of prior art, first panel between the adjacent storage tank unit is narrow, can't realize the cutting of above-mentioned Sealing strap and sealing again.
The symmetric crystallization plates of embodiment 2 24 * 3 non-mirror images
Referring to Figure 10 a to Figure 12 and Figure 15; Show a kind of high throughput protein crystalline 24 * 3 (promptly 4 * 6 * 3) symmetric crystallization of protein plate of non-mirror image that is used for; Its size conforms SBS standard; That is: meet the standard of ANSI/SBS1-2004 coverage area size, ANSI/SBS2-2004 height dimension and ANSI/SBS3-2004 bottom outside flange dimension and to ANSI/SBS4-2004: the hole site standard is improved, and the width of first panel 240 that each storage tank unit 20 has improves on the basis of SBS standard.
The symmetric crystallization of protein plate of 24 * 3 non-mirror images comprises crystallization plates main part 10, first panel, 240 total lengths of this main part be 124+/-1mm, total width be 82+/-1mm; The storage tank unit 20 that on crystallization plates main body 10, distributes, the length of each said storage tank unit 20 with 4 * 6 array formats be 16.0+/-0.25mm, width be 16.0+/-0.25mm; Each storage tank unit 20 forms a groove in first panel, 240 mid-ways; At this bottom portion of groove is second panel 250; At one first storage tank 220 of the recessed formation of second panel, 250 1 side positions; In second panel 250 of first storage tank, 220 offsides, be formed with three dropping baths 230; First panel 240 is connected through connection section 260 with second panel 250, and crystallization plates main part 10 has support portion 300 around it, and this support portion is by last support portion 320, support portion 340 and connect up and down that the support and connection portion 360 of support portion constitutes down.
Wherein, the distance between first panel 240 and second panel, 250 upper surfaces be 3.0+/-0.5mm; The width of first storage tank 220 be 8.0+/-0.5mm, the length of first storage tank 220 be 16.0+/-0.5mm, the degree of depth of first storage tank 220 be 8.25+/-0.5mm; The degree of depth of dropping bath 230 be 0.4+/-0.1mm, for upper end diameter R
OnBe 2.2+/-0.3, lower end diameter R
DownFor 1.7+/-the round table-like or degree of depth of 0.3mm be 1.0+/-0.2mm, diameter be 2.0+/-0.3mm closely hemispherical; Between the adjacent two storage tank unit 20 along the width of first panel 240 of crystallization plates length direction be 2.0+/-0.5mm, along the width of first panel 240 of crystallization plates width be 2.0+/-0.5mm; The total height of support portion 300 be 14.35+/-0.25mm, the height of wherein going up support portion 320 be 8.25mm+/-0.5mm, down the height of support portion 340 be 6.10+/-0.38mm and the width of support and connection portion 360 be 2.8+/-0.5mm.
It is 135 ° lead angle α and β that the end of first panel 240 on its width of crystallization plates has two respectively; And the last support portion 320 of crystallization plates, and support and connection portion 360 also have the lead angle α and the β of 135+/-10 ° respectively in corresponding position.
The length of first panel 240 between lead angle α, the β be 9.0+/-0.5mm.
Wherein, the width of first panel 240 between two adjacent storage tank unit 20 is about 2.0mm, its width much larger than crystallization of protein plate of the prior art (≤0.5mm).Therefore; Utilize 24 * 3 crystallization of protein plates of the present invention after each row or each row storage tank unit filling protein finish, to carry out each row or the unitary sealing of each row storage tank, and each the storage tank unit after the sealing can be realized sealing fully veritably with storage tank unit on every side.
In addition, in Figure 13, illustrated 24 * 6 crystallization of protein plate of (promptly 4 * 6 * 6).The shape and the distribution of the dropping bath 230 of other possible mirror images symmetric 48 * 4 (promptly 8 * 6 * 4) crystallization plates have been shown in Figure 14.Wherein 40 is the storage tank cell position and the application of sample center of 96 orifice plates among the ANSI/SBS4-2004; Can find out the application of sample center of not observing 96 orifice plates for the dropping bath 230 of 48 * 4 crystallization plates; Therefore during like scheme, whether the considered crystallization plates needs the robotization application of sample position of adherence to standard 96 orifice plates in design class in prompting.
The shape and the distribution of the dropping bath 230 of other the possible symmetric 24 * n of non-mirror image (n=5 or 6) crystallization plates have been shown in Figure 15.Wherein 40 is the storage tank cell position and the application of sample center of 96 orifice plates among the ANSI/SBS4-2004, can find out in each storage tank unit 20 to be the robotization application of sample position of part drop groove 230 adherence to standards 96 orifice plates.
Embodiment 3 utilizes the seat of the symmetric crystallization plates of 48 * 2 mirror images to drip the method crystallization of protein
Utilize the seat of high throughput protein crystallization plates to drip the method method of protein crystallization, may further comprise the steps:
1. the crystallizing pond liquid that contains buffer reagent, washing agent, precipitation agent or its combination with proper volume joins in first storage tank 220 in each storage tank unit 20; 8 road rifle sample adding devices wherein capable of using carry out manual application of sample, and automated multi-channel loading device also capable of using carries out automatic application of sample.
With the crystallizing pond liquid that contains buffer reagent, precipitation agent, washing agent or its combination of proper volume with to carry out the crystalline protein soln with certain proportion; For example volume ratio is 1: 1; Join in two dropping baths 220 in each storage tank unit 20, also can in dropping bath, add additive, washing agent or its combination as required simultaneously;
3. utilize Sealing strap 6 to cover on storage tank unit 20 first panel 240 all around; Each storage tank unit that will on the crystallization plates main body, distribute with array format seals, and has formed the internal medium of carrying out the running balance of crystallization of protein in each inside, storage tank unit;
4. crystallization plates is placed and carry out protein crystallization experiments under the temperature that is suitable for crystallization of protein.
Wherein, the protein soln in each storage tank unit in the different dropping bath can be identical or different, and the protein soln in the different dropping bath also can be identical or different in each different storage tank unit.
Alternatively, crystallization of protein process or result are monitored by robotization crystallization recording geometry, photographic camera, pick up camera or manual work.
Embodiment 4 utilizes the sessile drop method crystallization of protein of the symmetric crystallization plates of 48 * 2 mirror images
Similarly, (promptly 8 * 6 * 2) symmetric crystallization plates of mirror image carries out the sessile drop method crystallization of protein also can to utilize 48 * 2, may further comprise the steps:
1. the crystallizing pond liquid that contains buffer reagent, precipitation agent, washing agent or its combination with proper volume joins in first storage tank 220 in each storage tank unit 20; 8 road rifle sample adding devices wherein capable of using carry out manual application of sample, and automated multi-channel loading device also capable of using carries out automatic application of sample.
With proper volume with step 1 in identical pond liquid with to carry out the crystalline protein soln according to a certain percentage; For example 1: 2; And be added drop-wise on the shrouding film 6 according to the distribution situation of the storage tank unit 20 of crystallization plates 10, also can in above-mentioned drop, add additive, washing agent or its combination as required simultaneously;
3. shrouding film 6 is inverted and is covered on storage tank unit 20 first panel 240 all around; Utilize the shrouding film to be provided on the crystallization plates main body 10 sealing of each the storage tank unit 20 that distributes with array format, and in each storage tank unit the inner running balance environment of keeping crystallization of protein that forms;
4. crystallization plates is placed and carry out protein crystallization experiments under the temperature that is suitable for crystallization of protein.
Wherein, the protein soln in each storage tank unit in the different dropping bath can be identical or different, and the protein soln in the different dropping bath also can be identical or different in each different storage tank unit.
Alternatively, crystallization of protein process or result are monitored by robotization protein crystal growth recording geometry, photographic camera, pick up camera or manual work.
Embodiment 5 utilizes the method for protein crystallization of the symmetric crystallization plates of 24 * 3 non-mirror images
Concrete operations are similar with embodiment 3, and just a plurality of dropping baths 230 in each storage tank unit 20 are positioned at a side of first storage tank 220, and concrete crystallization of protein operation is similar with embodiment 3 or 4, can carry out manual application of sample and automatic application of sample.
The above is merely the preferred embodiments of the present invention, is not limited to the present invention, and for a person skilled in the art, the present invention can have various changes and variation.All within spirit of the present invention and principle, any modification of being done, be equal to replacement, improvement etc., all should be included within the protection domain of appending claims of the present invention.
Claims (14)
1. a crystallization of protein plate comprises crystallization plates main part (10), and the storage tank unit (20) that on said crystallization plates main body, distributes with array format; Each said storage tank unit (20) has first panel (240); Form a groove in the said first panel mid-way, said bottom portion of groove is second panel (250), in the said second panel mid-way or near recessed first storage tank (220) that forms in the position of a side; In second panel (250) of at least one side of said first storage tank (220), be formed with at least two dropping baths (230); Said first panel (240) is connected through connection section (260) with second panel (250), and said crystallization plates main part (10) has support portion (300) around it, wherein; Said crystallization plates is 48 orifice plates; Comprise crystallization plates main part (10), the total length of first panel (240) of said main part (10) is 120.5～125.5mm, and total width is 78.5～83.5mm; Wherein, the length of each said storage tank unit (20) is that 13.5～15.5mm, width are 6.5～7.5mm; Distance between said first panel (240) and second panel (250) upper surface is 2～8mm; The width of first storage tank (220) is 3.0～6.0mm, and the length of first storage tank (220) is 6.5～7.5mm, and the degree of depth of first storage tank (220) is 3.5～9.5mm; The upper end diameter R of dropping bath (230)
OnBe 3.0>=R
On>=1.5mm, lower end diameter R
DownBe 2.5>=R
Down>=0.5mm, the degree of depth of dropping bath (230) is 0.2～1.0mm; Width at storage tank unit (20) first panel (240) all around is 1.5～4.5mm, and the width of first panel (240) of periphery is 5.5～10.5mm in the storage tank unit (20) of said crystallization plates main part (10) most peripheral; The total height of said support portion (300) is 13.8～14.6mm.
2. crystallization plates according to claim 1; Wherein, Said at least two dropping baths (230) are a plurality of, in second panel (250) of the one or both sides of said first storage tank (220), are formed with a plurality of dropping baths (230), and the storage tank unit (20) that said array format distributes is 48; And said at least two dropping baths (230) are two, three, four, five or six.
3. according to the crystallization plates of claim 1, wherein, said dropping bath (230) independently is selected from the group that is made up of right cylinder, hemisphere, inverted Rotary-table or cone, cubes, rectangular parallelepiped with the shape of said first storage tank (220).
4. according to each described crystallization plates in the claim 1 to 3, wherein, said crystallization plates is processed by the high purity thermoplastics.
5. according to each described crystallization plates in the claim 1 to 3, wherein, said crystallization plates is sealed by Sealing strap or shrouding film.
6. according to each described crystallization plates in the claim 1 to 3; Wherein, Said crystallization plates is 48 orifice plates; In its size conforms SBS standard three, i.e. conformance with standard ANSI/SBS1-2004: coverage area size, ANSI/SBS2-2004: height dimension, ANSI/SBS3-2004: the bottom outside flange dimension, and to ANSI/SBS4-2004: improve the hole site.
7. according to each described crystallization plates in the claim 1 to 3, wherein, the volume of said first storage tank (220) is 0.04～1.4ml, and the volume of said dropping bath (230) is 0.2～7 μ l.
8. according to each described crystallization plates in the claim 1 to 3; Wherein, Said support portion (300) is by last support portion (320), following support portion (340) and connect support and connection portion (360) formation of support portion up and down; The wherein said height of going up support portion (320) is 6.1～12.5mm, the height of support portion (340) is 2.0～8.0mm down, and the width of the support and connection portion (360) of support portion is 1.0～4.0mm about connecting.
9. according to each described crystallization plates in the claim 1 to 3, wherein, first panel (240) of the said crystallization plates end on its width at least has two lead angle α and β respectively; Last support portion (320), and support and connection portion (360) also have lead angle α and β respectively corresponding to first panel (240) of said crystallization plates, said lead angle α and β are 135+/-10 °.
10. crystallization plates according to claim 4, said crystallization plates is processed by the high purity thermoplastics that is selected from polymeric amide, Vestolen PP 7052, PS, acrylonitrile-butadiene-styrene copolymer, polycarbonate, polymethylmethacrylate, styrene-acrylonitrile resin or gathers the cyclenes nitrile.
11. crystallization plates according to claim 4, said crystallization plates is processed by PS.
12. a method of utilizing each described crystallization plates of claim 1～11 to carry out crystallization of protein may further comprise the steps:
A. proper volume is joined in first storage tank (220) in each storage tank unit (20) by the crystallizing pond liquid (2) that is selected from buffer reagent, precipitation agent, washing agent or combinations thereof;
B. solution identical among proper volume and the step a is joined respectively at least one dropping bath (230) in each said storage tank unit (20) with carrying out the crystalline protein soln according to a certain percentage, also can in dropping bath, add additive, washing agent or its combination as required simultaneously;
C. utilize Sealing strap or shrouding film to cover on first panel (240) of said storage tank unit (20); The sealing of each the storage tank unit (20) that will be on said crystallization plates main body distributes with array format, and in each storage tank unit the inner running balance environment of keeping crystallization of protein that forms;
D. said crystallization plates is placed under the temperature of crystallization of protein and carry out crystallization of protein.
13. a method of utilizing each described crystallization plates of claim 1～11 to carry out crystallization of protein may further comprise the steps:
A. the crystallizing pond liquid (2) by being selected from buffer reagent, precipitation agent, washing agent or combinations thereof with proper volume joins in first storage tank (220) in each storage tank unit (20);
B. with proper volume with step a in identical solution be added drop-wise on Sealing strap or the shrouding film (6) according to the distribution situation of the storage tank unit (20) of said crystallization plates (10) according to a certain percentage with carrying out the crystalline protein soln, also can in above-mentioned drop, add additive, washing agent or its combination as required simultaneously;
C. said Sealing strap or shrouding film (6) are inverted and are covered on first panel (240) of said storage tank unit (20); Utilize said Sealing strap or shrouding film to be provided at the sealing that said crystallization plates main body (10) goes up each the storage tank unit (20) that distributes with array format, and form the running balance environment of keeping crystallization of protein in each inside, storage tank unit;
D. said crystallization plates is placed under the temperature of crystallization of protein and carry out protein crystallization experiments.
14. according to the method for claim 12 or 13 described crystallization of protein, said crystallization of protein process or result are monitored by robotization protein crystal growth recording geometry, photographic camera, pick up camera or manual work.
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| WO2010072026A1 (en) * | 2008-12-24 | 2010-07-01 | 北京大学 | Crystallization apparatus and method for crystallizing biological macromolecule and application thereof |
| CN102887939B (en) * | 2011-07-19 | 2015-08-19 | 江苏同庚电子科技有限公司 | The method of crystallization of protein is impelled with bio-vitric |
| CN104402968B (en) * | 2014-12-11 | 2018-01-30 | 西北工业大学 | A kind of method that crystallization of protein is carried out under gas permeability crystallization apparatus and the device |
| JP6848173B2 (en) * | 2015-10-09 | 2021-03-24 | 東レ株式会社 | Fiber-containing crystals, method for producing fiber-containing crystals, equipment for producing fiber-containing crystals, and chemical soaking equipment |
| JP2020502487A (en) * | 2016-10-28 | 2020-01-23 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Preparation of substances and analysis of solid properties |
| CN111100182B (en) * | 2019-12-17 | 2022-02-08 | 中国科学院上海高等研究院 | Driving device of disc type protein crystallization plate |
| CN111855718B (en) * | 2020-07-29 | 2023-05-12 | 中国科学院上海高等研究院 | Protein crystallization and crystal in-situ diffraction data acquisition device and acquisition method thereof |
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| WO2003087326A2 (en) * | 2002-04-10 | 2003-10-23 | Kendro Laboratory Products, Lp | Automated protein crystallization imaging |
| US6913732B2 (en) * | 2001-03-19 | 2005-07-05 | Corning Incorporated | Microplate for performing crystallography studies and methods for making and using such microplates |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6913732B2 (en) * | 2001-03-19 | 2005-07-05 | Corning Incorporated | Microplate for performing crystallography studies and methods for making and using such microplates |
| WO2003087326A2 (en) * | 2002-04-10 | 2003-10-23 | Kendro Laboratory Products, Lp | Automated protein crystallization imaging |
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