CN106404614A - Ice crystal observation method of ice structure protein - Google Patents
Ice crystal observation method of ice structure protein Download PDFInfo
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- CN106404614A CN106404614A CN201610726903.7A CN201610726903A CN106404614A CN 106404614 A CN106404614 A CN 106404614A CN 201610726903 A CN201610726903 A CN 201610726903A CN 106404614 A CN106404614 A CN 106404614A
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- 239000013078 crystal Substances 0.000 title claims abstract description 140
- 238000000034 method Methods 0.000 title claims abstract description 33
- 102000004169 proteins and genes Human genes 0.000 title abstract 6
- 108090000623 proteins and genes Proteins 0.000 title abstract 6
- 239000012460 protein solution Substances 0.000 claims abstract description 44
- 239000000243 solution Substances 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000008280 blood Substances 0.000 claims abstract description 18
- 210000004369 blood Anatomy 0.000 claims abstract description 18
- 230000008014 freezing Effects 0.000 claims abstract description 17
- 238000007710 freezing Methods 0.000 claims abstract description 17
- 230000003287 optical effect Effects 0.000 claims abstract description 13
- 239000000843 powder Substances 0.000 claims abstract description 7
- 101710172711 Structural protein Proteins 0.000 claims description 69
- 239000007788 liquid Substances 0.000 claims description 16
- 229910019142 PO4 Inorganic materials 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 7
- 239000010452 phosphate Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims 2
- 229910052698 phosphorus Inorganic materials 0.000 claims 2
- 239000011574 phosphorus Substances 0.000 claims 2
- 238000001514 detection method Methods 0.000 abstract description 3
- 230000007547 defect Effects 0.000 abstract 1
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 238000001179 sorption measurement Methods 0.000 abstract 1
- 241000219793 Trifolium Species 0.000 description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 22
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 15
- 229930006000 Sucrose Natural products 0.000 description 15
- 239000005720 sucrose Substances 0.000 description 15
- 241000209140 Triticum Species 0.000 description 14
- 235000021307 Triticum Nutrition 0.000 description 14
- 239000011780 sodium chloride Substances 0.000 description 11
- 230000000694 effects Effects 0.000 description 7
- 238000001000 micrograph Methods 0.000 description 5
- 238000001953 recrystallisation Methods 0.000 description 5
- 230000002528 anti-freeze Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 108010053481 Antifreeze Proteins Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101710082837 Ice-structuring protein Proteins 0.000 description 2
- 240000004658 Medicago sativa Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000005357 flat glass Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000006193 liquid solution Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000007711 solidification Methods 0.000 description 2
- 230000008023 solidification Effects 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101710123134 Ice-binding protein Proteins 0.000 description 1
- 235000010624 Medicago sativa Nutrition 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 101710107540 Type-2 ice-structuring protein Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
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- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to an ice crystal observation method of an ice structure protein, and aims to solve the problems that the ice crystal morphology of an ice structure protein solution can only be observed by a biological low temperature microscope, and the cost is high. The ice crystal observation method is as follows: 1, taking ice structure protein powder to dissolve in water or a phosphate buffer solution, centrifuging, taking 30muL of the solution to add dropwise into a groove of a blood counting plate, and putting the blood counting plate into a freezer for freezing; and 2, taking the frozen blood counting plate out, and transferring the blood counting plate under an ordinary optical microscope for observation and photographing by a 40*magnification lens. By use of the characteristics of ice crystal surface adsorption and ice crystal morphology change of the ice structure protein and combination of the ordinary optical microscope, the ice crystal morphology of the ice structure protein can be observed well, the method is simple, the problem that special equipment such as the low temperature microscope must be used for helping ice crystal observation is solved, and the defects of expensive equipment and complex detection method can be overcome. The method is suitable for the ice crystal observation of the ice structure protein.
Description
Technical field
The present invention relates to a kind of ice crystal observational technique of ice structural protein.
Background technology
Ice structural protein (ice structuring proteins, ISPs), is also called and does not freeze albumen, antifreeze protein
(antifreeze proteins, AFPs) is a class by some fish, insect, plant, fungi and bacterium is the extraneous ring of opposing
Polypeptide produced by the stress reaction of border, has and stops ice crystal from being formed, modifies ice crystal form and the ability of suppression recrystallization, it has
Certain heat stagnation activity, can be reduced the freezing point of solution, but the impact to fusing point is less with non-colligative property form, so that molten
Between the freezing point of liquid and fusing point, difference occurs.The distinctive property of ice structural protein ensures biology existence under cryogenic.
2006, the Ministry of Public Health of China announced ice structural protein and can be applied in chilled food as new food additive；Ice structure egg
Have the characteristics that adsorbing ice crystal surface changes ice crystal form, can make ice crystal produce various different shapes in vain, and not of uniform size, mainly
Form is quadrangle, pentagon, hexagon, rhombus, ellipse, fusiformis, bar-shaped and needle-like etc..
Prior art or changes typically using biological cryomicroscope technology to the observation of ice structural protein solution ice crystalline form state
The cryomicroscope technology entered.Cryomicroscope is come snap frozen protein solution using liquid ethane, instrument and its safeguard into
This is high, limits the popularization of cryomicroscope.Improved cryo-microscope technology, is to adopt liquid nitrogen to cool down its cold source system；
Or the cold and hot table apparatus of independent temperature control are installed additional on microscope；Or improve ordinary optical microscope for temperature control slide.
Using above technology observe ice structural protein solution ice crystal when, instrument and equipment is loaded down with trivial details limited, high cost.
Content of the invention
The present invention can only be by biological low temperature for the observation solving ice structural protein solution ice crystalline form state in prior art
Microscope is carried out, and equipment is loaded down with trivial details limited, and the problem of high cost is it is proposed that a kind of ice crystal observational technique of ice structural protein.
The ice crystal observational technique of ice structural protein of the present invention is carried out according to the following steps：
First, the preparation of sample
Take ice structural protein powder, be dissolved in water or PBS, be made into the ice knot that concentration is 10～30mg/mL
Structure protein solution, centrifugal treating, take 30 μ L of supernatant liquid to be placed on blood counting chamber with liquid-transfering gun, in -18 DEG C of refrigerator freezings 20～
30min；The pH of described PBS is 6.0～8.5, and phosphate concentration is 10～150mmol/L.
2nd, the observation of ice crystal
Take out the blood counting chamber of freezing from refrigerator, shift as under ordinary optical microscope in 5～10s, first
Under 10 × mirror, adjust light source, find object, treat that object is clear, turn 40 × sem observation, take pictures；Wherein, due to ice structure
Albumen is white slightly color mostly, and its solution is colourless or light formation, when observing ice crystal form, will suitably turn down light source
Brightness.
The present invention possesses following beneficial effect：
1st, ice structural protein has the characteristics that adsorbing ice crystal surface changes ice crystal form, can make ice crystal produce various not similar shapes
Shape, and not of uniform size, Main Morphology is quadrangle, pentagon, hexagon, rhombus, ellipse, fusiformis, bar-shaped and needle-like etc.；This
Invention using this feature and combines ordinary optical microscope, observed the ice crystal form of ice structural protein, method letter well
Just, ice crystal observation can be carried out using ordinary optical microscope, observing effect is good, solving must be by means of specialized instrument and equipment
As a difficult problem for cryo-microscope sem observation ice crystal, overcome instrument and equipment costliness, the complicated shortcoming of detection method；
2nd, the blood counting chamber adopting in the inventive method is made up of high-quality heavy sheet glass, and glass slide is thick, groove is deep, utilizes
Blood counting chamber observes the ice crystal form of liquid, just keeps the temperature of frozen liquid at room temperature, facilitates look at；
3rd, sodium chloride and sucrose are conventional cryoprotectors.The ice crystal Morphological Features of sodium chloride and sucrose solution are ice crystals
Little, quantity is many, and the ice crystal of water is very big, and these are all common ice crystal forms；Sodium chloride, sucrose is utilized when the present invention observes
Carrying out compareing with water is to verify that the form of ice structural protein ice crystal is rich and varied, and the modification to ice crystal form is notable,
It is also the checking that practicality of the present invention and ice crystal are observed with result simultaneously；
4th, the Activity of Antifreeze characteristic of ice structural protein includes modifying ice crystal form, quantity and recrystallization form, using this
Bright method can apply ordinary optical microscope that its Activity of Antifreeze is identified, and qualification result is true and reliable, accuracy
Identical with cryomicroscope；
5th, will be seen that the solidification process of liquid solution using ice crystal observational technique of the present invention, in the process the shape of ice crystal
Become and growth, the growth chracteristic of ice crystal and growth mechanism in the recrystallization freezing process of ice crystal, grasp the form of ice crystal and freeze
Process.
Brief description
Fig. 1 is for being in the ice crystal form 40x microphoto of pentagon or fusiformis in embodiment 1 clover ice structural protein solution；
Fig. 2 is cylindrical ice crystal form 40x microphoto in embodiment 1 clover ice structural protein solution；
Fig. 3 is to be in pentagonal ice crystal form 40x microphoto in embodiment 1 clover ice structural protein solution；
Fig. 4 is the ice crystal form 40x microphoto in embodiment 1 clover ice structural protein solution in quadrangle；
Fig. 5 is the micro- photograph of another 40x of the ice crystal form in embodiment 1 clover ice structural protein solution in quadrangle
Piece；
Fig. 6 is the ice crystal form 40x microphoto in embodiment 1 clover ice structural protein solution in triangle；
Fig. 7 is the ice crystal form 40x microphoto in embodiment 1 clover ice structural protein solution in hexagonal pyramid shape；
Fig. 8 is to be in quadrangle or the micro- photograph of pentagonal ice crystal form 40x in embodiment 1 clover ice structural protein solution
Piece；
Fig. 9 is hexagonal ice crystal form 40x microphoto in embodiment 1 clover ice structural protein solution；
Figure 10 is to be in quadrangle or the micro- photograph of hexagonal ice crystal form 40x in embodiment 1 clover ice structural protein solution
Piece；
Figure 11 is the ice crystal form 40x microphoto in embodiment 1 clover ice structural protein solution in hexagonal pyramid shape；
Figure 12 is rounded ice crystal form 40x microphoto in embodiment 1 water；
Figure 13 is the ice crystal form 40x microphoto in embodiment 1 water in fusiformis；
Figure 14 is rounded ice crystal form 40x microphoto in embodiment 1 sucrose solution；
Figure 15 is another 40x microphoto of rounded ice crystal form in embodiment 1 sucrose solution；
Figure 16 is rounded ice crystal form 40x microphoto in embodiment 1NaCl solution；
Figure 17 is the ice crystal form 40x microphoto in embodiment 1NaCl solution in fusiformis；
Figure 18 is another 40x microphoto of the ice crystal form in embodiment 1NaCl solution in fusiformis；
Figure 19 is the ice crystal form 40x microphoto in embodiment 1 cold ground winter wheat ice structural protein solution in water-drop-shaped；
Figure 20 is for being in that ice crystal form 40x of fusiformis or water-drop-shaped shows in embodiment 1 cold ground winter wheat ice structural protein solution
Micro- photo；
Figure 21 is the ice crystal form 40x microphoto in embodiment 1 cold ground winter wheat ice structural protein solution in fusiformis；
Figure 22 is rounded ice crystal form 40x microphoto in embodiment 1 cold ground winter wheat ice structural protein solution；
Figure 23 is in bar-shaped ice crystal form 40x microphoto in embodiment 1 cold ground winter wheat ice structural protein solution.
Specific embodiment：
Technical solution of the present invention is not limited to act specific embodiment set forth below, also includes between each specific embodiment
Arbitrarily reasonable combination.
Specific embodiment one：The ice crystal observational technique of present embodiment ice structural protein is carried out according to the following steps：
First, the preparation of sample
Take ice structural protein powder, be dissolved in water or PBS, be made into the ice knot that concentration is 10～30mg/mL
Structure protein solution, centrifugal treating, take 30 μ L of supernatant liquid to be placed on blood counting chamber with liquid-transfering gun, in -18 DEG C of refrigerator freezings 20～
30min；
2nd, the observation of ice crystal
Take out the blood counting chamber of freezing from refrigerator, shift as under ordinary optical microscope in 5～10s, first
Under 10 × mirror, adjust light source, find object, treat that object is clear, turn 40 × sem observation, take pictures；Wherein, due to ice structure
Albumen is white slightly color mostly, and its solution is colourless or light formation, when observing ice crystal form, will suitably turn down light source
Brightness.
Present embodiment possesses following beneficial effect：
1st, ice structural protein has the characteristics that adsorbing ice crystal surface changes ice crystal form, can make ice crystal produce various not similar shapes
Shape, and not of uniform size, Main Morphology is quadrangle, pentagon, hexagon, rhombus, ellipse, fusiformis, bar-shaped and needle-like etc.；This
Embodiment using this feature and combines ordinary optical microscope, observed the ice crystal form of ice structural protein well, side
Method is easy, can carry out ice crystal observation using ordinary optical microscope, observing effect is good, solving must be by means of instrumentation
A difficult problem for equipment such as cryo-microscope sem observation ice crystal, overcomes instrument and equipment costliness, the complicated shortcoming of detection method；
2nd, the blood counting chamber adopting in present embodiment method is made up of high-quality heavy sheet glass, and glass slide is thick, groove is deep,
Observe the ice crystal form of liquid using blood counting chamber, just keep the temperature of frozen liquid at room temperature, facilitate look at；
3rd, sodium chloride and sucrose are conventional cryoprotectors.The ice crystal Morphological Features of sodium chloride and sucrose solution are ice crystals
Little, quantity is many, and the ice crystal of water is very big, and these are all common ice crystal forms；Present embodiment observe when using sodium chloride,
It is to verify that the form of ice structural protein ice crystal is rich and varied that sucrose and water carry out compareing, and the modification to ice crystal form shows
Write, be also the checking that present embodiment practicality and ice crystal are observed with result simultaneously；
4th, the Activity of Antifreeze characteristic of ice structural protein includes modifying ice crystal form, quantity and recrystallization form, using this reality
Applying methods can apply ordinary optical microscope that its Activity of Antifreeze is identified, and qualification result is true and reliable, accurate
Really property is identical with cryomicroscope；
5th, the solidification process of liquid solution, ice crystal in the process is will be seen that using present embodiment ice crystal observational technique
Formation and growth, the growth chracteristic of ice crystal and growth mechanism in the recrystallization freezing process of ice crystal, grasp the form of ice crystal with
Freezing process.
Specific embodiment two：Present embodiment from unlike specific embodiment one：Phosphate described in step one delays
The pH rushing solution is 6.0～8.5, and phosphate concentration is 10～150mmol/L.Other steps and parameter and specific embodiment one
Identical.
Specific embodiment three：Present embodiment from unlike specific embodiment one or two：Phosphoric acid described in step one
The pH of salt buffer solution is 6.0～8.5, and phosphate concentration is 40mmol/L.Other steps and parameter and specific embodiment one
Or two is identical.
Specific embodiment four：Unlike one of present embodiment and specific embodiment one to three：Described in step
During centrifugal treating, the rotating speed of centrifuge is 4000～6000r/min.Other steps and parameter and specific embodiment one to three it
One is identical.
Specific embodiment five：Unlike one of present embodiment and specific embodiment one to four：Described in step
During centrifugal treating, centrifugation time is 10～15min.One of other steps and parameter and specific embodiment one to four are identical.
Specific embodiment six：Unlike one of present embodiment and specific embodiment one to five：Described in step
Take ice structural protein powder, be dissolved in PBS, be made into the ice structural protein solution that concentration is 30mg/mL, centrifugation
Process, take 30 μ L of supernatant liquid to be placed on blood counting chamber with liquid-transfering gun, in -18 DEG C of refrigerator freezing 20min.Other steps and parameter
Identical with one of specific embodiment one to five.
Embodiment 1
The ice crystal observational technique of the present embodiment clover ice structural protein is carried out according to the following steps：
First, the preparation of sample
Take clover ice structural protein powder, be dissolved in water or PBS, be made into the ice knot that concentration is 30mg/mL
Structure protein solution, is centrifuged 10min under 4000r/min, takes 30 μ L of supernatant liquid to be placed on blood counting chamber with liquid-transfering gun, in -18
DEG C refrigerator freezing 20min；The pH of described PBS is 8.0, and phosphate concentration is 40mmol/L；
2nd, the observation of ice crystal
Take out the blood counting chamber of freezing from refrigerator, shift as under ordinary optical microscope in 10s, first 10
Under × mirror, adjust light source, find object, treat that object is clear, turn 40 × sem observation, take pictures；Wherein, due to ice structural protein
It is white slightly color mostly, its solution is colourless or light formation, when observing ice crystal form, will suitably turn down light source bright
Degree.
Water intaking, the cold ground winter wheat ice structure egg of the NaCl solution of 30mg/mL, the sucrose solution of 30mg/mL and 30mg/mL
White solution as comparison, by the cold ground winter wheat of water, the NaCl solution of 30mg/mL, the sucrose solution of 30mg/mL and 30mg/mL
Ice structural protein solution is respectively placed in -18 DEG C of refrigerator freezing 20min；It is subsequently placed in 40 × optical microphotograph Microscopic observation and take pictures；
The ice crystal form 40x microphoto of clover ice structural protein solution manufactured in the present embodiment is as shown in Fig. 1～11；Its
In, Fig. 1 is for being in the ice crystal form 40x microphoto of pentagon or fusiformis in embodiment 1 clover ice structural protein solution；Fig. 2 is
Cylindrical ice crystal form 40x microphoto in embodiment 1 clover ice structural protein solution；Fig. 3 is embodiment 1 clover ice structure
It is in pentagonal ice crystal form 40x microphoto in protein solution；Fig. 4 is to be in four in embodiment 1 clover ice structural protein solution
The ice crystal form 40x microphoto of side shape；Fig. 5 is the ice crystal form in embodiment 1 clover ice structural protein solution in quadrangle
Another 40x microphoto；Fig. 6 is that in embodiment 1 clover ice structural protein solution, ice crystal form 40x in triangle is micro-
Photo；Fig. 7 is the ice crystal form 40x microphoto in embodiment 1 clover ice structural protein solution in hexagonal pyramid shape；Fig. 8 is real
Apply in example 1 clover ice structural protein solution is in quadrangle or pentagonal ice crystal form 40x microphoto；Fig. 9 is embodiment 1 lucerne
Hexagonal ice crystal form 40x microphoto in Mu ice structural protein solution；Figure 10 is that embodiment 1 clover ice structural protein is molten
It is in quadrangle or hexagonal ice crystal form 40x microphoto in liquid；Figure 11 is to be in embodiment 1 clover ice structural protein solution
The ice crystal form 40x microphoto of hexagonal pyramid shape；From Fig. 1～11, the ice of ice crystal volume vs' water of alfalfa solution
But crystal amasss wants little bigger than sucrose solution ice crystal volume, but ice crystal form is enriched, and is partly the cylindricality similar to NaCl ice crystal
Ice crystal；Part is in fusiformis, quadrangle, the ice crystal of the hexagonal pyramid shape of pentagonal ice crystal form, hexagon ice crystal and solid；
The ice crystal form 40x microphoto of the water that the present embodiment obtains is as shown in Figure 12～13；Wherein, Figure 12 is embodiment
Rounded ice crystal form 40x microphoto in 1 water；Figure 13 is the micro- photograph of ice crystal form 40x in embodiment 1 water in fusiformis
Piece；From Figure 12～13, the ice crystal form of water is larger circle, and arrangement is closely；
The ice crystal form 40x microphoto of the sucrose solution that the present embodiment obtains is as shown in Figure 14～15；Wherein, Tu14Wei
Rounded ice crystal form 40x microphoto in embodiment 1 sucrose solution；Figure 15 is rounded in embodiment 1 sucrose solution
Another 40x microphoto of ice crystal form；From Figure 14～15, the ice crystal volume of volume vs' water of sucrose solution ice crystal
Much little, and form is single, and the ice crystal plesiomorphism of water, mostly circular；
The ice crystal form 40x microphoto of the NaCl solution that the present embodiment obtains is as shown in Figure 16～18；Wherein, Tu16Wei
Rounded ice crystal form 40x microphoto in embodiment 1NaCl solution；Figure 17 is in fusiformis in embodiment 1NaCl solution
Ice crystal form 40x microphoto；Figure 18 is the micro- photograph of another 40x of the ice crystal form in embodiment 1NaCl solution in fusiformis
Piece；From Figure 16～18, the ice crystal small volume of NaCl solution, quantity is more, but form is more single；
Molten ice crystal form 40x microphoto such as Figure 19～23 institute of cold ground winter wheat ice structural protein that the present embodiment obtains
Show；Wherein, Figure 19 is the ice crystal form 40x microphoto in embodiment 1 cold ground winter wheat ice structural protein solution in water-drop-shaped；
Figure 20 is for being in the ice crystal form 40x microphoto of fusiformis or water-drop-shaped in embodiment 1 cold ground winter wheat ice structural protein solution；Figure
21 is the ice crystal form 40x microphoto in embodiment 1 cold ground winter wheat ice structural protein solution in fusiformis；Figure 22 is embodiment
Rounded ice crystal form 40x microphoto in 1 cold ground winter wheat ice structural protein solution；Figure 23 is that the embodiment 1 cold ground winter is little
It is in bar-shaped ice crystal form 40x microphoto in wheat ice structural protein solution.From Figure 19～23, ground winter wheat ice structure of trembling with fear
The ice crystal form of protein solution is in water-drop-shaped and circle mostly, is evenly distributed, polygon, stub shape or aciculiform also.
Claims (6)
1. a kind of ice crystal observational technique of ice structural protein is it is characterised in that this is carried out according to the following steps：
First, the preparation of sample
Take ice structural protein powder, be dissolved in water or PBS, be made into the ice structure egg that concentration is 10～30mg/mL
White solution, centrifugal treating, take 30 μ L of supernatant liquid to be placed on blood counting chamber with liquid-transfering gun, in -18 DEG C of refrigerator freezings 20～
30min；
2nd, the observation of ice crystal
Take out the blood counting chamber of freezing from refrigerator, shift as under ordinary optical microscope in 5～10s, first 10
Under × mirror, adjust light source, find object, treat that object is clear, turn 40 × sem observation, take pictures.
2. a kind of ice structural protein according to claim 1 ice crystal observational technique it is characterised in that：Phosphorus described in step one
The pH of hydrochlorate cushioning liquid is 6.0～8.5, and phosphate concentration is 10～150mmol/L.
3. a kind of ice structural protein according to claim 1 ice crystal observational technique it is characterised in that：Phosphorus described in step one
The pH of hydrochlorate cushioning liquid is 6.0～8.5, and phosphate concentration is 40mmol/L.
4. a kind of ice structural protein according to claim 1 ice crystal observational technique it is characterised in that：Described in step one from
When the heart is processed, the rotating speed of centrifuge is 4000～6000r/min.
5. a kind of ice structural protein according to claim 1 ice crystal observational technique it is characterised in that：Described in step one from
When the heart is processed, centrifugation time is 10～15min.
6. a kind of ice structural protein according to claim 1 ice crystal observational technique it is characterised in that：Take described in step one
Ice structural protein powder, is dissolved in PBS, is made into the ice structural protein solution that concentration is 30mg/mL, at centrifugation
Reason, takes 30 μ L of supernatant liquid to be placed on blood counting chamber with liquid-transfering gun, in -18 DEG C of refrigerator freezing 20min.
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| CN201610726903.7A CN106404614A (en) | 2016-08-25 | 2016-08-25 | Ice crystal observation method of ice structure protein |
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| CN201610726903.7A CN106404614A (en) | 2016-08-25 | 2016-08-25 | Ice crystal observation method of ice structure protein |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108444876A (en) * | 2018-03-09 | 2018-08-24 | 国家纳米科学中心 | A kind of assay method of nano grain surface adhesion protein ligand state |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1214615A (en) * | 1996-02-09 | 1999-04-21 | 雀巢制品公司 | Ice crystal growth inhibiting agents |
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