CN108196057A - A kind of method for observing large biological molecule distribution and application - Google Patents
A kind of method for observing large biological molecule distribution and application Download PDFInfo
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- CN108196057A CN108196057A CN201711407601.4A CN201711407601A CN108196057A CN 108196057 A CN108196057 A CN 108196057A CN 201711407601 A CN201711407601 A CN 201711407601A CN 108196057 A CN108196057 A CN 108196057A
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- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000009826 distribution Methods 0.000 title claims abstract description 25
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- 239000003086 colorant Substances 0.000 claims abstract description 3
- 230000002163 immunogen Effects 0.000 claims abstract description 3
- 239000003550 marker Substances 0.000 claims abstract description 3
- 239000012224 working solution Substances 0.000 claims description 82
- 239000007853 buffer solution Substances 0.000 claims description 63
- 239000007788 liquid Substances 0.000 claims description 21
- YJHDFAAFYNRKQE-YHPRVSEPSA-L disodium;5-[[4-anilino-6-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-2-[(e)-2-[4-[[4-anilino-6-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulfonatophenyl]ethenyl]benzenesulfonate Chemical compound [Na+].[Na+].N=1C(NC=2C=C(C(\C=C\C=3C(=CC(NC=4N=C(N=C(NC=5C=CC=CC=5)N=4)N(CCO)CCO)=CC=3)S([O-])(=O)=O)=CC=2)S([O-])(=O)=O)=NC(N(CCO)CCO)=NC=1NC1=CC=CC=C1 YJHDFAAFYNRKQE-YHPRVSEPSA-L 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000012103 Alexa Fluor 488 Substances 0.000 claims description 16
- 241000283074 Equus asinus Species 0.000 claims description 16
- 150000004676 glycans Chemical class 0.000 claims description 16
- 239000012114 Alexa Fluor 647 Substances 0.000 claims description 14
- 238000004140 cleaning Methods 0.000 claims description 14
- 239000000975 dye Substances 0.000 claims description 10
- 238000010166 immunofluorescence Methods 0.000 claims description 10
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- 229920001282 polysaccharide Polymers 0.000 claims description 8
- 239000005017 polysaccharide Substances 0.000 claims description 8
- 238000004043 dyeing Methods 0.000 claims description 6
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- 238000002965 ELISA Methods 0.000 claims 1
- 238000003125 immunofluorescent labeling Methods 0.000 abstract description 4
- 238000002372 labelling Methods 0.000 abstract description 3
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- 241000219194 Arabidopsis Species 0.000 description 19
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- 241000196324 Embryophyta Species 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 6
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- 238000010186 staining Methods 0.000 description 4
- 229920002230 Pectic acid Polymers 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 239000010318 polygalacturonic acid Substances 0.000 description 3
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- 238000010998 test method Methods 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
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- 235000003351 Brassica cretica Nutrition 0.000 description 1
- 235000003343 Brassica rupestris Nutrition 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses a kind of method for observing large biological molecule distribution and applications, belong to biological immune detection technique field.The method of present invention observation large biological molecule distribution, it is to make large biological molecule different in sample respectively by different fluorescence or dye marker using improved immunofluorescence staining, according to the difference of label, the distribution situation of different large biological molecules is observed simultaneously under the microscope；The improved immunofluorescence staining is：It selects different large biological molecules the primary antibody label of different immunogenes or selects different large biological molecules identical immunogene but carry the primary antibody label of different labels；Different colours is selected different large biological molecules to mark and develop the color with the secondary antibody of a corresponding anti-binding respectively.The method of the present invention can make the different fluorescence of different molecular labelings, can directly observe the distribution of various biomolecules, be conducive to find intermolecular interaction.
Description
Technical field
The invention belongs to biological immune detection technique fields, and in particular to it is a kind of observe large biological molecule distribution method and
Using.
Background technology
China is traditional large agricultural country, and for crop growth, the research of develop etc. is always biology and agriculture
Learn the emphasis and hot spot of research.The process of crop growthing development, in essence, important feature and tissue exactly in plant
Building process is exactly to include polysaccharide from molecular level, protein and other interaction, interactional mistake
Journey.Crops are carried out with the key of basic research, first consists in these large biological molecules and to be distributed in plant and the sight of structure
Examine process.
Immunofluorescent Antibody labelling technique is that fluorescein is formed immune mark chemically with specific antibody covalent bond
Remember antibody, and according to the principle of Ag-Ab specific bond, antigen is marked with fluorescein, and utilize fluorescence microscope
Specific fluorescent is observed, so as to intuitively observe distributed process of the antigen in plant.Due to spy of the antigen-antibody effect with identification
The opposite sex, and no particular requirement is formed for antigen, therefore, which has in terms of crop plant structure composition is parsed
Wide applicability.
At present, the application of fluorescence antibody labelling technique is ripe day by day, but traditional detection method determines this
The limitation of detection, i.e., observed by immunofluorescence, can only show the Germ distribution of single structure, obtained relevant information
It measures less than normal and single, the interaction between structure and Relative distribution intuitively can not be observed on the whole, this pole
The earth limits the application of this technology.
Invention content
For problems of the prior art, technical scheme is as follows：
A kind of method for observing large biological molecule distribution, life different in sample is made using improved immunofluorescence staining
Object macromolecular by different fluorescence or dye marker, according to the difference of label, observes different biologies simultaneously under the microscope respectively
The distribution situation of macromolecular；
The improved immunofluorescence staining is：The primary antibody of different immunogenes is selected different large biological molecules to mark
Or it selects different large biological molecules identical immunogene and carries the primary antibody label of different labels；To big point different of biologies
Selection different colours are marked and are developed the color with the secondary antibody of a corresponding anti-binding son respectively.
On the basis of said program, the large biological molecule is carbohydrate.
On the basis of said program, the primary antibody is JIM series antibodies, in LM series antibodies, CCRC series antibodies
A kind of and CBM series antibodies in a kind of combination.
On the basis of said program, the secondary antibody is Alexa Fluor line fluorescent labelled antibodies.
On the basis of said program, complete or part of the sample for histotomy or with excellent permeability is planted
Object tissue.
On the basis of said program, the method for the observation large biological molecule distribution, step is as follows：
1) selection of material
Select histotomy or the complete or Activities of Some Plants tissue with excellent permeability；
2) it closes
Confining liquid closing sample thermostatted water yawing moves 1h, removes confining liquid, PE buffer solution for cleaning.
3) primary antibody is immune labeled
A. corresponding primary antibody is selected according to the polysaccharide type of required detection, the primary antibody of selection is configured to primary antibody working solution,
Sample is combined；
B. primary antibody working solution, PE buffer solution for cleaning are removed；
4) secondary antibody is immune labeled
1. JIM/LM+CBM is as primary antibody
A. the antibody working solution of the anti-His of mouse is added in into sample, thermostatted water yawing moves 0.5h；
B. remove the antibody working solution of mouse anti-His, PE buffer solution for cleaning three times, 10 minutes every time；
C. the anti-rat Ab of donkey of the Alexa Fluor-488 of preparation labels and Alexa Fluor-647 are marked
The mixing secondary antibody working solution of donkey anti-mouse antibody, is combined sample, thermostatted water yawing moves 0.5h；
D. remove secondary antibody working solution, PE buffer solution for cleaning three times, 10 minutes every time；
2. CCRC+CBM is as primary antibody
The donkey anti-mouse antibody working solution of a.Alexa Fluor-488 labels is combined sample, and thermostatted water yawing moves
0.5h；
B. remove working solution, PE buffer solution for cleaning three times, 10 minutes every time；
C. the antibody working solution of the anti-His of mouse is combined sample, and thermostatted water yawing moves 0.5h；
D. remove working solution, PE buffer solution for cleaning three times, 10 minutes every time；
The donkey anti-mouse antibody working solution of e.Alexa Fluor-647 labels is combined sample, and thermostatted water yawing moves
0.5h；
F. remove working solution, PE buffer solution for cleaning three times, 10 minutes every time；
5) it develops the color
The complete sample of above-mentioned processing adds Calcofluor White working solutions to dye sample 15 minutes；
6) image acquisition
Sample after colour developing is added dropwise PE buffer solutions to observe under Laser Scanning Confocal Microscope.
On the basis of said program, it is 37 DEG C that the thermostatted water yawing, which moves, 50rpm.
On the basis of said program, the primary antibody is combined into 37 DEG C, and 50rpm thermostatted water yawings move 0.8h.
On the basis of said program, the primary antibody working solution and secondary antibody working solution be by each antibody by 1: 1 volume ratio
It mixes.
A kind of kit for observing large biological molecule distribution, resists including confining liquid, PE buffer solutions, primary antibody, secondary antibody, mouse
The antibody and developing solution of His；
The primary antibody is JIM series antibodies, LM series antibodies, one kind in CCRC series antibodies and CBM series antibodies
In a kind of combination；
The secondary antibody is Alexa Fluor line fluorescent labelled antibodies；
The developing solution is Calcofluor White working solutions.
Beneficial effects of the present invention
1st, for immunofluorescence technique of the invention compared with original monoclonal antibody immunofluorescence dyeing, the method for the present invention is glimmering using difference
Light marks different polysaccharide molecules respectively, can directly observe the Relative distribution between two kinds of polysaccharide, conducive to find polysaccharide between can
It existing can interact.
Though the 2, the method for the present invention is that two kinds of polysaccharide structures are carried out while dyed based on two kinds of antibody, it is carried out at the same time dye
The sugared structure of color depends on the secondary antibody type that can mutually distinguish, and theoretically, secondary antibody type is more, while the sugared structure dyed is just
It is abundanter.The method of the present invention will lay the first stone to develop while carrying out immune labeled method for a variety of sugared structures later.
Description of the drawings
Fig. 1 embodiment 1JIM5+CBM3a antibody colour developing result overall diagram (left side develop the color simultaneously for two kinds of antibody as a result, in
Between be JIM5 individually colour developing as a result, the right is CBM3a individually develops the color result)；
Fig. 2 embodiment 1JIM5+CBM3a antibody colour developing result Local map (left side develop the color simultaneously for two kinds of antibody as a result, in
Between be JIM5 individually colour developing as a result, the right is CBM3a individually develops the color result)；
Fig. 3 embodiment 2JIM7+CBM3a antibody colour developing result Local map (left side develop the color simultaneously for two kinds of antibody as a result, in
Between be JIM7 individually colour developing as a result, the right is CBM3a individually develops the color result)；
Fig. 4 embodiment 3CCRC-M36+CBM3a antibody colour developing result Local map (develops the color knot on the left side simultaneously for two kinds of antibody
Fruit, centre be CCRC-M36 individually colour developing as a result, the right is CBM3a individually develops the color result)；
Fig. 5 embodiment 4CCRC-M38+CBM3a antibody colour developing result Local map (develops the color knot on the left side simultaneously for two kinds of antibody
Fruit, centre be CCRC-M38 individually colour developing as a result, the right is CBM3a individually develops the color result)；
Fig. 6 comparative example 1CCRC-M38+CBM3a antibody colour developing result Local map (develops the color knot on the left side simultaneously for two kinds of antibody
Fruit, centre be CCRC-M38 individually colour developing as a result, the right is CBM3a individually develops the color result)；
Fig. 7 comparative example 2CCRC-M38+CBM3a antibody colour developing result Local map (develops the color knot on the left side simultaneously for two kinds of antibody
Fruit, centre be CCRC-M38 individually colour developing as a result, the right is CBM3a individually develops the color result)；
Fig. 8 comparative example 3CCRC-M38+CBM3a antibody colour developing result Local map (develops the color knot on the left side simultaneously for two kinds of antibody
Fruit, centre be CCRC-M38 individually colour developing as a result, the right is CBM3a individually develops the color result).
Specific embodiment
Used term in the present invention, it is unless otherwise specified, generally usual with those of ordinary skill in the art
The meaning of understanding.
With reference to specific embodiment, and with reference to the data further detailed description present invention.Following embodiment only be
It illustrates the present invention rather than limits the scope of the invention in any way.
The preparation of reagent
1) PE buffer solutions：PBS+EDTA buffer solutions
1.64g NaCl(140mM)、0.04g KCl、0.04g KH2PO4、0.716g Na2HPO4、8μL 0.25M
EDTA, pH value 7.4.
2) confining liquid：
Skimmed milk power, a concentration of 0.03g/mL of skimmed milk power are added in PE buffer solutions.
3) primary antibody working solution
JIM, LM and CBM series antibody are bought from Leeds, England university PlantProbes website (http://
www.plantprobes.net/index.php#contact)；
CCRC series antibodies are bought from U.S. Carbosource websites (http://www.carbosource.net/)；
Primary antibody working solution is configured according to product description with primary antibody product using the confining liquid being configured.
4) the anti-His antibody working solution of mouse：
Stoste is using PE buffer solutions with 1:1000 volume ratios are configured.
5) secondary antibody working solution
Intermediate antibody and the purchase of secondary antibody Immunofluorescent Antibody with His labels match silent winged generation certainly, and you are scientific and technological
(https://www.thermofisher.com)；
Primary antibody working solution is configured according to product description with secondary antibody product using the confining liquid being configured.
6) Calcofluor White working solutions：
Calcofluor White dye liquor stostes are bought from Sigma-Aldrich websites (product identification：18909,
Www.sigmaaldrich.com) with PE buffer solutions with volume ratio 1:5 configurations.
Test material
The sample is not affected by the histotomy of destruction for large biological molecule or has the complete or portion of excellent permeability
Divide plant tissue.
The present invention is selected for the kind skin mucilaginous substance of arabidopsis, it is intended to be explored and be improved original technology step.The present invention's
So it is object to select arabidopsis kind skin mucilaginous substance, the mucilaginous design feature of arabidopsis kind skin is allowed for.Arabidopsis kind skin
Mucilaginous substance is in the atomization of arabidopsis seed coat cell, and one layer of epidermal cell of kind skin most surface can one layer of secreting outside
Water-soluble colloid substance is wrapped in seed coat surface, and after dry seed meets water imbibition, these mucilaginous substances discharge to be formed at once
Gelatin matter is simultaneously coated with entire seed completely.By carrying out Multi-layer technology to kind of skin mucilaginous substance and carrying out sugared composition analysis discovery, intend
The southern mucilaginous the Nomenclature Composition and Structure of Complexes of mustard kind skin is similar to plant cell wall, containing the polysaccharide such as cellulose, hemicellulose, pectin into
Point and a series of protein and metal ion.But unlike cell wall, kind skin mucilaginous substance does not have rigid structure, just
Transparent state is presented under normal situation, therefore, after handling kind of skin mucilaginous substance using immunofluorescence technique, immunofluorescence can be direct
By immunofluorescence microscopy, without carrying out histotomy first, this is the exploitation of new immunologic detection method, is saved
Plenty of time.
Embodiment 1
JIM5+CBM3a is selected as primary antibody
The same polygalacturonic acid glycan structures (low esterification HG) of the low esterification of JIM5 specific recognitions,
The cellulosic structure of CBM3a specific recognitions crystallization；
1) material selection
Arabidopsis seed (specifically study be arabidopsis seed kind skin mucilaginous substance).
2) it closes
C. sample is closed using the confining liquid of 200 μ L.
D. sample is placed in constant incubator and keeps the temperature 1 hour in 37 DEG C, horizontal shake is carried out with 50rpm rates.
E. with liquid-transfering gun remove confining liquid, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
3) primary antibody is immune labeled
A. JIM5 and CBM3a antibody is bought, antibody is diluted according to antibody specification, the antibody after two kinds of dilutions is isometric
It is uniformly mixed, is configured to mixing primary antibody working solution, 50 μ L is taken to be combined sample.
B. sample is placed in constant incubator and keeps the temperature 0.8 hour in 37 DEG C, horizontal shake is carried out with 50rpm rates.
C. with liquid-transfering gun remove primary antibody working solution, using PE buffer solutions by sample clean three times, with liquid-transfering gun absorb PE delay
Fliud flushing.
4) secondary antibody is immune labeled
A. the antibody working solution of the anti-His of mouse is configured first, 50 μ L working solutions is taken to be combined sample, sample is placed
0.5 hour is kept the temperature in 37 DEG C in constant incubator, horizontal shake is carried out with 50rpm rates.
B. with liquid-transfering gun remove working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
C. the anti-rat Ab of donkey (with reference to JIM5 primary antibodies) and Alexa Fluor- Alexa Fluor-488 marked
The donkey anti-mouse antibody of 647 labels dilutes to specifications (with reference to CBM3a-His tibody complex), is mixedly configured into equal volume
Secondary antibody working solution is mixed, 50 μ L working solutions is taken to be combined sample, sample is placed in constant incubator in 37 DEG C of heat preservations
0.5 hour, horizontal shake is carried out with 50rpm rates.
D. with liquid-transfering gun remove secondary antibody working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, use liquid relief
Rifle absorbs PE buffer solutions.
5) it develops the color
Using PE buffer solutions configuration Calcofluor White working solutions (1: 5), 50 this working solution of μ L is taken to dye sample
15 minutes.
6) image acquisition
It a. will treated that sample adds drips PE buffer solutions.
B. sample is focused on using 4 times of object lens of Lycra TCS SP8 Laser Scanning Confocal Microscopes.
C. according to the type of selected fluorescein, 405nm (Calcofluor White fluorescent excitings), 488nm are opened
(AlexaFluor-488 fluorescent excitings) and 638nm (Alexa Fluor-647 fluorescent excitings) fluorescence, manual focus
After shoot corresponding fluorescence photo.
Result of the test is as shown in Figure 1, 2：Fig. 1 is overall diagram, and Fig. 2 is partial enlarged view, wherein,
Red fluorescence in intermediate picture, be Alexa Fluor-488 fluorescence, in the present embodiment, represent JIM5 for
The colour developing result of low esterification HG structures.
Image to right is green fluorescence, be Alexa Fluor-647 fluorescence, in the present embodiment, represent CBM3a for
Crystallize the colour developing of cellulose.
Image to left is the fusion figure after each map overlay, and blue-fluorescence therein is Calcofluor White to β-Portugal
The unspecific staining of glycan, as the Background From Layer of immunofluorescence dyeing, red fluorescence represents JIM5 for low esterification HG
The colour developing of structure, green fluorescence represent colour developings of the CBM3a for crystallization cellulose, the red fluorescence portion Chong Die with green fluorescence
Divide display yellow fluorescence (circle irises out part in figure), representing low esterification HG structures, there are possible phases with crystallization cellulose
Interaction.
Embodiment 2
JIM7+CBM3a is selected as primary antibody
The same polygalacturonic acid glycan structures (high esterification HG) of the high esterification of JIM7 specific recognitions,
1) material selection
Arabidopsis seed (specifically study be arabidopsis seed kind skin mucilaginous substance).
2) it closes
C. sample is closed using the confining liquid of 200 μ L.
D. sample is placed in constant incubator and keeps the temperature 1 hour in 37 DEG C, horizontal shake is carried out with 50rpm rates.
E. with liquid-transfering gun remove confining liquid, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
3) primary antibody is immune labeled
A. JIM7 and CBM3a antibody is bought, antibody is diluted according to antibody specification, the antibody after two kinds of dilutions is isometric
It is uniformly mixed, is configured to mixing primary antibody working solution, 50 μ L is taken to be combined sample.
B. sample is placed in constant incubator and keeps the temperature 0.8 hour in 37 DEG C, horizontal shake is carried out with 50rpm rates.
C. with liquid-transfering gun remove primary antibody working solution, using PE buffer solutions by sample clean three times, with liquid-transfering gun absorb PE delay
Fliud flushing.
Second step：Secondary antibody is immune labeled
A. the antibody working solution of the anti-His of mouse is configured first, 50 μ L working solutions is taken to be combined sample, sample is placed
0.5 hour is kept the temperature in 37 DEG C in constant incubator, horizontal shake is carried out with 50rpm rates.
B. with liquid-transfering gun remove working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
C. the anti-rat Ab of donkey (with reference to JIM7 primary antibodies) and Alexa Fluor- Alexa Fluor-488 marked
The donkey anti-mouse antibody of 647 labels dilutes to specifications (with reference to CBM3a-His tibody complex), is mixedly configured into equal volume
Secondary antibody working solution is mixed, 50 μ L working solutions is taken to be combined sample, sample is placed in constant incubator in 37 DEG C of heat preservations
0.5 hour, horizontal shake is carried out with 50rpm rates.
D. with liquid-transfering gun remove secondary antibody working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, use liquid relief
Rifle absorbs PE buffer solutions.
5) it develops the color
Using PE buffer solutions configuration Calcofluor White working solutions (1: 5), 50 this working solution of μ L is taken to dye sample
15 minutes.
6) image acquisition
It a. will treated that sample adds drips PE buffer solutions.
B. sample is focused on using 4 times of object lens of Lycra TCS SP8 Laser Scanning Confocal Microscopes.
C. according to the type of selected fluorescein, 405nm (Calcofluor White fluorescent excitings), 488nm are opened
(AlexaFluor-488 fluorescent excitings) and 638nm (Alexa Fluor-647 fluorescent excitings) fluorescence, manual focus
After shoot corresponding fluorescence photo.
Result of the test is as shown in Figure 3：
Red fluorescence in intermediate picture is the fluorescence of Alexa Fluor-488, in the present embodiment, represents JIM7 in height
The colour developing result of esterification HG structures.
Image to right is green fluorescence, be Alexa Fluor-647 fluorescence, in the present embodiment, represent CBM3a for
Crystallize the colour developing of cellulose.
Image to left is the fusion figure after each map overlay, and blue-fluorescence therein is Calcofluor White to β-Portugal
The unspecific staining of glycan, as the Background From Layer of immunofluorescence dyeing, red fluorescence represents JIM7 for high esterification HG
The colour developing of structure, green fluorescence represent colour developings of the CBM3a for crystallization cellulose, the red fluorescence portion Chong Die with green fluorescence
Divide display yellow fluorescence, representing high esterification HG structures, there are possible interactions with crystallization cellulose.
Embodiment 3
CCRC-M36+CBM3a is selected as primary antibody
The backbone structure of CCRC-M36 specific recognitions phammogalacturonane (RG-I),
1) material selection
Arabidopsis seed (specifically study be arabidopsis seed kind skin mucilaginous substance).
2) it closes
C. sample is closed using the confining liquid of 200 μ L.
D. sample is placed in constant incubator and keeps the temperature 1 hour in 37 DEG C, horizontal shake is carried out with 50rpm rates.
E. with liquid-transfering gun remove confining liquid, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
3) primary antibody is immune labeled
A. CCRC-M36 and CBM3a antibody is bought, antibody, the antibody after two kinds of dilutions etc. are diluted according to antibody specification
Volume mixture is uniform, is configured to mixing primary antibody working solution, 50 μ L is taken to be combined sample.
B. sample is placed in constant incubator and keeps the temperature 0.8 hour in 37 DEG C, horizontal shake is carried out with 50rpm rates.
C. with liquid-transfering gun remove primary antibody working solution, using PE buffer solutions by sample clean three times, with liquid-transfering gun absorb PE delay
Fliud flushing.
4) secondary antibody is immune labeled
A. the donkey anti-mouse antibody (this step combines CCRC-M36 antibody) Alexa Fluor-488 marked is to specifications
Working solution is configured to, 50 μ L working solutions is taken to be combined sample, sample is placed in constant incubator and keeps the temperature 0.5 in 37 DEG C
Hour, horizontal shake is carried out with 50rpm rates.
B. with liquid-transfering gun remove working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
C. the antibody working solution of the anti-His of configuration mouse, takes 50 μ L working solutions to be combined sample, sample is placed on perseverance
0.5 hour is kept the temperature in 37 DEG C in warm incubator, horizontal shake is carried out with 50rpm rates.
D. with liquid-transfering gun remove working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
E. the donkey anti-mouse antibody (this step combines CBM3a-His tibody complex) that Alexa Fluor-647 are marked is pressed
Book is configured to hybrid working liquid as directed, and 50 μ L working solutions is taken to be combined sample, sample is placed in constant incubator
0.5 hour is kept the temperature in 37 DEG C, horizontal shake is carried out with 50rpm rates.
F. with liquid-transfering gun remove working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
5) it develops the color
Using PE buffer solutions configuration Calcofluor White working solutions (1: 5), 50 this working solution of μ L is taken to dye sample
15 minutes.
6) image acquisition
It a. will treated that sample adds drips PE buffer solutions.
B. sample is focused on using 4 times of object lens of Lycra TCS SP8 Laser Scanning Confocal Microscopes.
C. according to the type of selected fluorescein, 405nm (Calcofluor White fluorescent excitings), 488nm are opened
(AlexaFluor-488 fluorescent excitings) and 638nm (Alexa Fluor-647 fluorescent excitings) fluorescence, manual focus
After shoot corresponding fluorescence photo.
Result of the test is as shown in Figure 4：
Red fluorescence in intermediate picture is the fluorescence of Alexa Fluor-488, in the present embodiment, represents CCRC-M36
In the colour developing result of RG-I backbone structures.
Image to right is green fluorescence, be Alexa Fluor-647 fluorescence, in the present embodiment, represent CBM3a for
Crystallize the colour developing of cellulose.
Image to left is the fusion figure after each map overlay, and blue-fluorescence therein is Calcofluor White to β-Portugal
The unspecific staining of glycan, as the Background From Layer of immunofluorescence dyeing, red fluorescence represents CCRC-M36 for RG-I master
The colour developing of chain structure, green fluorescence represent colour developings of the CBM3a for crystallization cellulose, and red fluorescence is Chong Die with green fluorescence
Part shows yellow fluorescence, and representing RG-I backbone structures, there are possible interactions with crystallization cellulose.
Embodiment 4
CCRC-M38+CBM3a is selected as primary antibody
The same polygalacturonic acid glycan structures (non-esterification HG) of the non-esterification of CCRC-M38 specific recognitions
1) material selection
Arabidopsis seed (specifically study be arabidopsis seed kind skin mucilaginous substance).
2) it closes
C. sample is closed using the confining liquid of 200 μ L.
D. sample is placed in constant incubator and keeps the temperature 1 hour in 37 DEG C, horizontal shake is carried out with 50rpm rates.
E. with liquid-transfering gun remove confining liquid, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
3) primary antibody is immune labeled
A. CCRC-M36 and CBM3a antibody is bought, antibody, the antibody after two kinds of dilutions etc. are diluted according to antibody specification
Volume mixture is uniform, is configured to mixing primary antibody working solution, 50 μ L is taken to be combined sample.
B. sample is placed in constant incubator and keeps the temperature 0.8 hour in 37 DEG C, horizontal shake is carried out with 50rpm rates.
C. with liquid-transfering gun remove primary antibody working solution, using PE buffer solutions by sample clean three times, with liquid-transfering gun absorb PE delay
Fliud flushing.
4) secondary antibody is immune labeled
A. the donkey anti-mouse antibody (this step combines CCRC-M38 antibody) Alexa Fluor-488 marked is to specifications
Working solution is configured to, 50 μ L working solutions is taken to be combined sample, sample is placed in constant incubator and keeps the temperature 0.5 in 37 DEG C
Hour, horizontal shake is carried out with 50rpm rates.
B. with liquid-transfering gun remove working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
C. the antibody working solution of the anti-His of configuration mouse, takes 50 μ L working solutions to be combined sample, sample is placed on perseverance
0.5 hour is kept the temperature in 37 DEG C in warm incubator, horizontal shake is carried out with 50rpm rates..
D. with liquid-transfering gun remove working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
E. the donkey anti-mouse antibody (this step combines CBM3a-His tibody complex) that Alexa Fluor-647 are marked is pressed
Book is configured to hybrid working liquid as directed, and 50 μ L working solutions is taken to be combined sample, sample is placed in constant incubator
0.5 hour is kept the temperature in 37 DEG C, horizontal shake is carried out with 50rpm rates.
F. with liquid-transfering gun remove working solution, using PE buffer solutions by sample clean three times, 10 minutes every time, inhaled with liquid-transfering gun
Except PE buffer solutions.
5) it develops the color
Using PE buffer solutions configuration Calcofluor White working solutions (1: 5), 50 this working solution of μ L is taken to dye sample
15 minutes.
6) image acquisition
It a. will treated that sample adds drips PE buffer solutions.
B. sample is focused on using 4 times of object lens of Lycra TCS SP8 Laser Scanning Confocal Microscopes.
C. according to the type of selected fluorescein, 405nm (Calcofluor White fluorescent excitings), 488nm are opened
(AlexaFluor-488 fluorescent excitings) and 638nm (Alexa Fluor-647 fluorescent excitings) fluorescence,
Result of the test is as shown in Figure 5：
Red fluorescence in intermediate picture is the fluorescence of Alexa Fluor-488, in the present embodiment, represents CCRC-M38
In the colour developing result of non-esterification HG structures.
Image to right is green fluorescence, be Alexa Fluor-647 fluorescence, in the present embodiment, represent CBM3a for
Crystallize the colour developing of cellulose.
Image to left is the fusion figure after each map overlay, and blue-fluorescence therein is Calcofluor White to β-Portugal
The unspecific staining of glycan, as the Background From Layer of immunofluorescence dyeing, red fluorescence represents CCRC-M38 for non-methyl esters
Change the colour developing of HG structures, green fluorescence represents colour developings of the CBM3a for crystallization cellulose, and red fluorescence is Chong Die with green fluorescence
Part show yellow fluorescence (circle irises out part in figure), representing that non-esterification HG structures exist with crystallization cellulose may
Interaction.
Comparative example 1
Antibody selected as CCRC-M38+CBM3a
Material selection：Arabidopsis seed (specifically study be arabidopsis seed kind skin mucilaginous substance).
Test method：Selection of speed 100rpm during constant temperature shakes, other steps are the same as embodiment 4.
As a result：As shown in fig. 6, the red fluorescence of colour developings of the CCRC-M38 to non-esterification HG structures is represented in intermediate picture
Colour developing is very weak, and discontinuous, and Image to right represents CBM3a and divergent shape is presented for the green fluorescence of crystallization cellulose colour developing
State.
Comparative example 2
Antibody selected as CCRC-M38+CBM3a
Material selection：Arabidopsis seed (specifically study be arabidopsis seed kind skin mucilaginous substance).
Test method：Temperature selects 28 DEG C during constant temperature shakes, other steps are the same as embodiment 4.
As a result：As shown in fig. 7, the red fluorescence of colour developings of the CCRC-M38 to non-esterification HG structures is represented in intermediate picture
Develop the color weak, Image to right represent CBM3a for crystallization cellulose colour developing green fluorescence it is relatively weak, some parts are significantly not
Coloring.The two lap, i.e. yellow fluorescence partial disappearance.
Comparative example 3
Antibody selected as CCRC-M38+CBM3a
Material selection：Arabidopsis seed (specifically study be arabidopsis seed kind skin mucilaginous substance).
Test method：Selection of time 1.5h during constant temperature shakes, other steps are the same as embodiment 4.
As a result：As shown in figure 8, the red fluorescence of colour developings of the CCRC-M38 to non-esterification HG structures is represented in intermediate picture
Colour developing completely disappears, and it is too strong for the green fluorescent coloration of crystallization cellulose colour developing that Image to right represents CBM3a.
The above described is only a preferred embodiment of the present invention, being not that the invention has other forms of limitations, appoint
What those skilled in the art changed or be modified as possibly also with the technology contents of the disclosure above equivalent variations etc.
Imitate embodiment.But it is every without departing from technical solution of the present invention content, technical spirit according to the present invention is to above example institute
Any simple modification, equivalent variations and the remodeling made still fall within the protection domain of technical solution of the present invention.
Claims (10)
- A kind of 1. method for observing large biological molecule distribution, it is characterised in that：Made in sample using improved immunofluorescence dyeing Different large biological molecules by different fluorescence or dye marker, according to the difference of label, is observed simultaneously under the microscope respectively The distribution situation of different large biological molecules；The improved enzyme linked immunosorbent assay is：Different large biological molecule is selected different immunogenes primary antibody label or It selects different large biological molecules identical immunogene and carries the primary antibody label of different labels；To different large biological molecule point It different colours label and Xuan Ze not develop the color with the secondary antibody of a corresponding anti-binding.
- 2. the method for observation large biological molecule distribution according to claim 1, it is characterised in that：The large biological molecule is plants Object polysaccharide.
- 3. the method for observation large biological molecule distribution according to claim 1 or claim 2, it is characterised in that：The primary antibody is JIM A kind of combination in a kind of and CBM series antibodies in series antibody, LM series antibodies, CCRC series antibodies.
- 4. the method for observation large biological molecule distribution according to claim 3, it is characterised in that：The secondary antibody is Alexa Fluor line fluorescent labelled antibodies.
- 5. the method for observation large biological molecule distribution according to claims 1 to 4, it is characterised in that：The sample is group Knit slice or the complete or Activities of Some Plants tissue with excellent permeability.
- 6. the method for observation large biological molecule distribution according to claim 5, it is characterised in that：Step is as follows：1) selection of materialSelect histotomy or the complete or Activities of Some Plants tissue with excellent permeability；2) it closesConfining liquid closing sample thermostatted water yawing moves 1h, removes confining liquid, PE buffer solution for cleaning.3) primary antibody is immune labeledA. corresponding primary antibody is selected according to the polysaccharide type of required detection, the primary antibody of selection is configured to primary antibody working solution, to sample Product are combined；B. primary antibody working solution, PE buffer solution for cleaning are removed；4) secondary antibody is immune labeled1. JIM/LM+CBM is as primary antibodyA. the antibody working solution of the anti-His of mouse is added in into sample, thermostatted water yawing moves 0.5h；B. remove the antibody working solution of mouse anti-His, PE buffer solution for cleaning three times, 10 minutes every time；C. the donkey of the anti-rat Ab of donkey of the Alexa Fluor-488 of preparation labels and Alexa Fluor-647 labels is resisted The mixing secondary antibody working solution of mouse antibodies, is combined sample, thermostatted water yawing moves 0.5h；D. remove secondary antibody working solution, PE buffer solution for cleaning three times, 10 minutes every time；2. CCRC+CBM is as primary antibodyThe donkey anti-mouse antibody working solution of a.Alexa Fluor-488 labels is combined sample, and thermostatted water yawing moves 0.5h；B. remove working solution, PE buffer solution for cleaning three times, 10 minutes every time；C. the antibody working solution of the anti-His of mouse is combined sample, and thermostatted water yawing moves 0.5h；D. remove working solution, PE buffer solution for cleaning three times, 10 minutes every time；The donkey anti-mouse antibody working solution of e.Alexa Fluor-647 labels is combined sample, and thermostatted water yawing moves 0.5h；F. remove working solution, PE buffer solution for cleaning three times, 10 minutes every time；5) it develops the colorThe complete sample of above-mentioned processing adds Calcofluor White working solutions to dye sample 15 minutes；6) image acquisitionSample after colour developing is added dropwise PE buffer solutions to observe under Laser Scanning Confocal Microscope.
- 7. the method for observation large biological molecule distribution according to claim 6, it is characterised in that：The thermostatted water yawing moves It is 37 DEG C, 50rpm.
- 8. the method for observation large biological molecule distribution according to claim 6, it is characterised in that：The primary antibody is combined into 37 DEG C, 50rpm thermostatted water yawings move 0.8h.
- 9. the method for observation large biological molecule distribution according to claim 6, it is characterised in that：The primary antibody working solution and two Anti- working solution is to be mixed by each antibody by 1: 1 volume ratio.
- 10. a kind of kit for observing large biological molecule distribution, it is characterised in that：Including confining liquid, PE buffer solutions, primary antibody, two Anti-, the anti-His of mouse antibody and developing solution；The primary antibody is JIM series antibodies, in a kind of and CBM series antibodies in LM series antibodies, CCRC series antibodies A kind of combination；The secondary antibody is Alexa Fluor line fluorescent labelled antibodies；The developing solution is Calcofluor White working solutions.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102072959A (en) * | 2010-11-16 | 2011-05-25 | 武汉大学 | Method for simultaneously labelling collagen type IV, macrophage and neovascularisation of tumors |
| CN103063849A (en) * | 2012-12-28 | 2013-04-24 | 武汉大学 | A method for simultaneous detection of cancer-associated fibroblasts and expressed proteins thereof |
| CN103926408A (en) * | 2014-04-30 | 2014-07-16 | 天津医科大学肿瘤医院 | Kit for forecasting NSCLC (non-small-cell lung cancer) patient prognosis and application of osteopontin acting as specific marker |
| CN103994911A (en) * | 2014-03-19 | 2014-08-20 | 中国人民解放军第四军医大学 | Immunofluorescence double labeling method based on same species source first antibody |
| CN104237504A (en) * | 2014-09-19 | 2014-12-24 | 汕头大学医学院 | Immunofluorescence method and kit for pathological autopsy of anaphylactic shock |
| CN105067813A (en) * | 2015-07-23 | 2015-11-18 | 丁晓昆 | Method for rapidly detecting T-synthase activity |
-
2017
- 2017-12-22 CN CN201711407601.4A patent/CN108196057B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102072959A (en) * | 2010-11-16 | 2011-05-25 | 武汉大学 | Method for simultaneously labelling collagen type IV, macrophage and neovascularisation of tumors |
| CN103063849A (en) * | 2012-12-28 | 2013-04-24 | 武汉大学 | A method for simultaneous detection of cancer-associated fibroblasts and expressed proteins thereof |
| CN103994911A (en) * | 2014-03-19 | 2014-08-20 | 中国人民解放军第四军医大学 | Immunofluorescence double labeling method based on same species source first antibody |
| CN103926408A (en) * | 2014-04-30 | 2014-07-16 | 天津医科大学肿瘤医院 | Kit for forecasting NSCLC (non-small-cell lung cancer) patient prognosis and application of osteopontin acting as specific marker |
| CN104237504A (en) * | 2014-09-19 | 2014-12-24 | 汕头大学医学院 | Immunofluorescence method and kit for pathological autopsy of anaphylactic shock |
| CN105067813A (en) * | 2015-07-23 | 2015-11-18 | 丁晓昆 | Method for rapidly detecting T-synthase activity |
Non-Patent Citations (2)
| Title |
|---|
| YINGPING CAO ET AL: "Cell wall polysaccharide distribution in Miscanthus lutarioriparius stem using immuno-detection", 《PLANT CELL REP》 * |
| 宋兴辉等: "脑组织双重免疫荧光标记方法介绍", 《电子显微学报》 * |
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