CN105859705A - Fluorescence labelled probe as well as preparation method and application thereof in protein labeling - Google Patents
Fluorescence labelled probe as well as preparation method and application thereof in protein labeling Download PDFInfo
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- CN105859705A CN105859705A CN201610381017.5A CN201610381017A CN105859705A CN 105859705 A CN105859705 A CN 105859705A CN 201610381017 A CN201610381017 A CN 201610381017A CN 105859705 A CN105859705 A CN 105859705A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- 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"
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
- C09K2211/1048—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with oxygen
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- 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 fluorescence labelled probe as well as a preparation method and application thereof in protein labeling. An organic synthesis method is adopted, and an oxadiazole heterocycle with fluorescent properties and multiple heteroatoms is directly introduced to the bottom ring of a rhodamine B skeleton structure. The introduced oxadiazole heterocycle serves as a second fluorophore for expanding the spectroscopic properties of rhodamine fluorescence probes; and by introducing multiple heteroatoms, the sites forming hydrogen bonds are increased, and the action with amino acids in the protein is promoted. In a compound RH-MCl, one side of the oxadiazole cycle is substituted by rhodamine B, and the other side is substituted by chloromethyl. The invention also provides a method for gel electrophoresis fluorescence labeling of protein using the fluorescence probe. The compound RH-MCl has good water solubility, and the fluorescence labeling method for protein has the advantages of easiness, short time, high sensitivity, good reproducibility, good labeling background, good compatibility, safety in use, low cost and the like.
Description
The present invention is in Tianjin application foundation and cutting edge technology research plan (Contract NO: 14JCYBJC23900)
Carry out under subsidy.
Technical field
The present invention relates to preparation and the protein fluorescence labeling method of a kind of fluorescence labeling probe, belong to chemical biology neck
Territory.
Background technology
Fluorescence probe is in fields such as molecular biology, microbiology, biochemistry, analytical chemistry, chemical industry and medical industries
Also have and apply widely.Its development simultaneously is closely related with subjects such as biotechnology, environmental science, diagnostic medicines.Along with
The development of the related disciplines such as chemistry, physics and biotechnology and progress, be born the most relevant to fluorescence probe
Technology, these technology have expanded range of application and the depth of investigation of fluorescence probe greatly so that fluorescence probe is increasingly becoming existing
For the strong research means of new technical field.The kind of fluorescent molecular probe is also carried by the variation day by day of these recent studies on means
Go out higher requirement.
In life science field, be unableing to do without the base substance albumen Quality Research to life, it is right also just to be unable to do without
The detection technique of protein, the most in recent years, along with developing rapidly of proteomics, makes our the detection skill to protein
Art has had higher requirement.Gel electrophoresis is analysis complex proteins mixture, analyzes a certain protein relative amount, detects egg
White matter purity, the biochemical analysis method of assessment protein molecular weight.In electrophoresis system, band dyeing is an important step
Suddenly.After electrophoresis, the method for protein staining mainly has Coomassie brilliant blue, silver staining, negative staining etc. at present.But protein is carried out
Fluorescently-labeled report is the rarest.Develop fluorescence probe easy to operate, low cost, higher sensitivity and be used for protein
Mark has great importance.
Summary of the invention
First purpose of the present invention is to provide a kind of fluorescence probe with molecular formula I and preparation method thereof.
Second object of the present invention is to utilize this fluorescence probe that gel electrophoresis of protein is carried out fluorescently-labeled method,
It is simple that the method has method of operating, and the used time is few, and expense is low, it is simple to observes, the advantage such as detection.
Third object of the present invention is to disclose chloromethyl diazole rhodamine compound in protein fluorescence mark side
The application in face.
For achieving the above object, the invention discloses following technology contents:
Having the chloromethyl rhodamine diazole (being called for short RH-MCl) of structural formula I, chemical name is 2-chloromethyl-5-[rhodamine
B-9'-(2''-benzoyl) base]-1,3,4-diazole；
The invention discloses the preparation method of chloromethyl diazole Rhodamine fluorescent probe:
(1) preparation of rhodamine hydrazides:
Take absolute ethyl alcohol (100ml) to be placed in the flask of 250ml, rhodamine B (5g, 10.4mmol) is dissolved in ethanol.Then
80% hydrazine hydrate (8ml, 133mmol) is slowly added dropwise at ambient temperature in mixed system.By mixture after dropping
It is heated to reflux.When solution is gradually become clarification by mulberry, and TLC monitors, reacting the complete room temperature that is cooled to, it is molten that decompression removes part
Agent, pour in frozen water, have precipitation to produce, suction filtration, obtain dark yellow solid.
(2) preparation of chloromethyl diazole rhodamine:
Monoxone 0.76g (0.1g, 1.1mmol) and POCl3 (5 ml) are mixed, is heated to reflux 0.5-1 hour, afterwards,
Being dividedly in some parts rhodamine B hydrazides (0.5g, 1.1mmol), TLC monitors, and question response is cooled to after completing, and adds a small amount of dichloromethane
Alkane is to room temperature and pours in mixture of ice and water, and regulation pH is 8, has to inorganic layer solution colour ratio with dichloromethane extraction crude product
Till when machine layer solution colour is deep, merge organic layer, wash with 5ml × 2 saturated aqueous common salt, separate organic phase.Use anhydrous slufuric acid
Magnesium is dried and stands overnight, and leaches drier, and vacuum rotary steam removes excess of solvent, gained crude product methylene chloride/methanol=20:
1, pillar layer separation, obtain target compound RH-MCl.
The present invention further discloses chloromethyl diazole rhodamine, as fluorescence probe, protein is carried out fluorescence labeling
Application.Wherein protein s DS-PAGE gel electrophoresis labeling method:
(1) weigh 0.055gRH-MCl to be dissolved in the acetum that 100ml volume ratio is 2%, be configured to the spy that concentration is 1000uM
Pin mother liquor, is diluted to concentration by this mother liquor with the acetum of 2% and is respectively the probe solution of 5uM, 10uM, 20uM, 40uM.
(2) SDS-PAGE protein example gel electrophoresis, Protein standards Premixed are carried out according to common method
Protein Marker (Low) is purchased from Takara company.
(3) take 4 pieces fixing after protein example gel be respectively placed in fluorescence probe concentration be 5uM, 10 uM, 20 uM,
The solution of 40uM marks 20min, 80 rpm decolorization swinging tables are washed with deionized 2hr, take out gel at ultraviolet gel
Detecting under imager, detection wavelength is 302nm.Take pictures (Fig. 5).Analyze through software Gel-pro Analyzer and draw optimal spy
Pin label concentration (Fig. 6).
(4) separately take 4 pieces fixing after protein example gel be placed in the solution that fluorescence probe concentration is 20uM, mark respectively
Note 5min, 10 min, 20 min, 40min, be washed with deionized 2hr on 80 rpm decolorization swinging tables, takes out gel in ultraviolet
Detecting under gel imaging instrument, detection wavelength is 302nm.Take pictures (Fig. 7).Analyze through software Gel-pro Analyzer and draw spy
The pin optimum mark time (Fig. 8).
(5) the most separately take 4 pieces fixing after protein example gel be placed in the solution that probe is 20uM, after mark 20min,
After being washed with deionized 1hr, 2hr, 3hr, 4hr on 80 rpm decolorization swinging tables respectively, take out gel at ultraviolet gel imaging
Detecting under instrument, detection wavelength is 302nm.Take pictures (Fig. 9).Analyze through software Gel-pro Analyzer and show that probe is most preferably washed
Wash the time (Figure 10).
The method of the described conventional SDS-PAGE gel electrophoresis of protein of above-mentioned steps (2) is as follows:
1. glass plate, sample comb, Spacer washing agent are cleaned, rinse for several times with distilled water, ethanol, dry；Two
Add Spacer between block glass plate, install glass plate；
2. between glass plate, record separation gel solution, cover one layer of distilled water immediately, stand 30 min and make separation gel polymerisation in solution,
Then being inclined by upper strata distilled water, filter paper blots, and records concentration sol solution, inserts sample comb, stands 30 min and make concentration peptization
Liquid is polymerized；
3. electrophoresis system is installed, adds Tris-glycine electrode buffer, gel lane after polymerisation adds protein
Sample, is adjusted to 100V by electrophoresis apparatus voltage, starts electrophoresis, when bromophenol blue indicator migrates to bottom separation gel, closes power supply
Stop electrophoresis；
4. unload offset plate, take out protein example gel, with distilled water washing by soaking 3min, be placed in fixer and fix 5min,
3min is rinsed again with distilled water；Standby.
Agents useful for same compound method is as follows:
1. the preparation of 5x sample buffer: take respectively Tris-HCl (pH6.8) 0.6ml of 1mol/L, 50% glycerine 5ml, 10%
SDS 2ml, mercaptoethanol 0.5ml, 1% bromophenol blue 1ml, distilled water 0.9ml, make 5x sample buffer 10ml, 4 DEG C or-20
DEG C refrigerator preserves.
2. the preparation of separation gel solution: take distilled water 1.6ml, acrylamide/methylene diacrylamide (29:1) respectively
30% stoste 2.0ml, 1.5M Tris-HCl(pH8.8) buffer solution 1.3ml, 10% SDS 50ul, 10% ammonium persulfate 50ul,
TEMED 2ul, is configured to the separation gel solution 5ml of 12%.
3. the preparation of sol solution is concentrated: take distilled water 1.4ml, acrylamide/methylene diacrylamide (29:1) respectively
30% stoste 0.33ml, 1M Tris-HCl(pH6.8) 0.26ml, 10% SDS 20ul, 10% ammonium persulfate 20ul, tetraethyl second
Diamines (TEMED) 2ul, is configured to the concentration sol solution 2ml of 5%.
4. the preparation of Tris-glycine electrode buffer (pH8.3): weigh Tris 6.0g, glycine 28.8g respectively,
Add distilled water about 900ml, after adjusting pH8.3, be settled to 1000ml with distilled water.4 DEG C of preservations, before use dilution 10 times.
5. fixer is containing 40% ethanol and 10% second aqueous acid.
Ultraviolet gel imaging instrument involved in above method is that Beijing 61 bio tech ltd produces, model
WD-9413B。
The result explanation of detection:
(1) the optimal concentration and probe concentration marked protein s DS-PAGE gel electrophoresis with RH-MCl is 20uM；Said by Fig. 5, Fig. 6
Bright.
(2) the band OD value marked protein s DS-PAGE gel electrophoresis with RH-MCl increases with the prolongation of mark time
Greatly, optimum mark time range is 20-40min；Fig. 7, Fig. 8 explanation.
(3), after protein s DS-PAGE gel electrophoresis being marked with RH-MCl, use distilled water to protein example gel
Washing by soaking can obtain experimental result in 2 hours.Fig. 9, Figure 10 explanation.
Fluorescence labeling probe disclosed by the invention and preparation method thereof and to the fluorescence labeling method of protein and existing skill
Art compares having the active effect that of being had
(1) optimizing rhodamine B fluorescent dye performance, novel fluorescence probe introduces azoles heterocycle, makes rhodamine probe skeleton
Introduce N, O hetero atom while there is Bichromophore, it is provided that hydrogen binding sites, improve fluorescence probe quantum yield and water-soluble；
Introduce benzyl position chlorine atom, make probe be beneficial to and the amino acid effect in protein.
(2) novel RH-MCl fluorescence probe sensitiveness is strong, and efficiency is high, can make SDS-PAGE protein electrophorese in the short period
Band is clearly prone to detection.Comparing with rhodamine B, FITC and additive method, required concentration and probe concentration is low, time-consumingly few, marks effect
Good.
Accompanying drawing illustrates:
The nucleus magnetic hydrogen spectrum of Fig. 1, RH-MCl；
The nuclear-magnetism carbon spectrum of Fig. 2, RH-MCl；
The flight time mass spectrum of Fig. 3, RH-MCl；
Fig. 4, RH-MCl(concentration is 20uM) fluorescence spectrum；
Protein gel imaging (a:5uM, b:10uM, c:20uM, d:40uM) after Fig. 5, different RH-MCl concentration markers；
Fig. 6, the different RH-MCl concentration impact on OD value；
Fig. 7, non-isolabeling time protein gel imaging (a:5min, b:10min, c:20min, d:40min)；
The impact on OD value of Fig. 8, the non-isolabeling time；
Fig. 9, different wash time protein gel imaging (a:1hr, b:2hr, c:3hr, d:4hr)；
Figure 10, the different wash time impact on OD value；
Protein gel imaging (a:RH-MCl, b: rhodamine B, c:FITC, d: examine horse after Figure 11, different probe (dyestuff) mark
This light blue)；
The gel imaging of Figure 12, certainly purification WspR protein.
Figure 13, the structural formula of chloromethyl rhodamine diazole.
Detailed description of the invention
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.It addition, embodiment is interpreted as illustrative, and the unrestricted present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
On the premise of invention spirit and scope, the various changes carrying out the material component in these embodiments and consumption or change are also
Belong to protection scope of the present invention, institute in monoxone therein, POCl3, rhodamine B and SDS-PAGE protein gel
It is commercially available with reagent.
Embodiment 1
The synthesis of fluorescence probe chloromethyl diazole rhodamine compound R H-MCl fluorescence probe:
(1) preparation of rhodamine hydrazides:
Take absolute ethyl alcohol (100ml) to be placed in the flask of 250ml, rhodamine B (5g, 10.4mmol) is dissolved in ethanol.Then
80% hydrazine hydrate (8ml, 133mmol) is slowly added dropwise at ambient temperature in mixed system.By mixture after dropping
It is heated to reflux.When solution is gradually become clarification by mulberry, and TLC monitors, reacting the complete room temperature that is cooled to, it is molten that decompression removes part
Agent, pour in frozen water, have precipitation to produce, suction filtration, obtain dark yellow solid.
(2) preparation of chloromethyl diazole rhodamine:
Monoxone 0.76g (0.1g, 1.1mmol) and POCl3 (5 ml) are mixed, is heated to reflux 0.5-1 hour, afterwards,
Being dividedly in some parts rhodamine B hydrazides (0.5g, 1.1mmol), TLC monitors, and question response is cooled to after completing, and adds a small amount of dichloromethane
Alkane is to room temperature and pours in mixture of ice and water, and regulation pH is 8, has to inorganic layer solution colour ratio with dichloromethane extraction crude product
Till when machine layer solution colour is deep, merge organic layer, wash with 5ml × 2 saturated aqueous common salt, separate organic phase.Use anhydrous slufuric acid
Magnesium is dried and stands overnight, and leaches drier, and vacuum rotary steam removes excess of solvent, gained crude product methylene chloride/methanol=20:
1, pillar layer separation, obtain target compound RH-MCl.Fusing point: 228-230 DEG C；Structure determines the nuclear-magnetism hydrogen seeing Fig. 1, RH-MCl
Spectrum；The nuclear-magnetism carbon spectrum of Fig. 2, RH-MCl.
Embodiment 2
Select several conventional protein fluorescence probe and dyestuff, such as: fluorescein isothiocynate (FITC), rhodamine B, coomassie
Light blue, is configured to and the solution of RH-MCl same concentrations (20uM), according to above-mentioned protein s DS-PAGE gel electrophoresis mark
Method, will fixing after Premixed Protein Marker (Low) protein gel be respectively placed in 20uM RH-MCl,
FITC, rhodamine B, Coomassie Brillant Blue solution mark (or dyeing) 20min, 80 rpm decolorization swinging tables are washed with deionized water
Wash 2hr(Coomassie brilliant blue and use decolouring washing at night), take out gel and detect under ultraviolet gel imaging instrument, detection wavelength is
302nm, the detection wavelength of Coomassie brilliant blue is visible ray.Take pictures (Figure 11).
Solution allocation used is as follows:
1. Coomassie brilliant blue G250 dyeing liquor: weigh Coomassie brilliant blue G250 100mg, is dissolved in 200ml distilled water, slowly adds
Enter 70% cross chloric acid 7.5ml, finally supply water to 250ml, stir 1 hour, aperture Filter paper filtering.
2. destainer: measure glacial acetic acid 75mL, distilled water 875mL and methyl alcohol 50mL mixing.
3. FITC probe solution: weigh FITC 50mg, adds 4ml distilled water, is configured to the mother liquor of 32mM, dilute with distilled water
Release to 20uM.
4. rhodamine B probe solution: weigh 2.395g rhodamine B, adds 5ml distilled water and makes it dissolve, and is configured to 1mM female
Liquid, is diluted to 20uM with distilled water.
Testing result illustrates:
Protein marker is through SDS-PAGE gel electrophoresis, FITC fluorescence probe, after marking, band mays be seen indistinctly；By examining horse
After this light blue G-250 dyeing, background coloration is relatively deep, and band is unintelligible；After being marked by rhodamine B, fluorescence intensity is less than RH-MCl.
It follows that identical in probe (or dyestuff) concentration, dyeing time is identical, wash time identical under conditions of, involved by the present invention
And RH-MCl fluorescence probe the mark effect of Premixed Protein Marker (Low) protein gel is better than other
Three kinds of fluorescence probes (or dyestuff).Figure 11 explanation.
Embodiment 3
RH-MCl probe is used for from the WspR protein qualitative detection purified.
(1) after the 100ml pseudomonas aeruginosa induction containing recombinant expression plasmid, centrifugal 5000g × 5min, abandons supernatant, receives
Obtain thalline, resuspended with the B/W buffer solution of 10ml precooling.
(2) re-suspension liquid is in the most ultrasonically treated, until sample no longer thickness, 4 DEG C of centrifugal 14000g × 30min, takes supernatant
Liquid, with after 0.45 micron membrane filter suction filtration as sample liquid.
(3) sample liquid is carried out ni-sepharose purification, obtain from the WspR protein purified.
(4) the WspR protein purified is grasped according to above-mentioned protein s DS-PAGE gel electrophoresis labeling method
Make, be placed in the RH-MCl of 20uM mark 20min, 80 rpm decolorization swinging tables are washed with deionized 2hr, take out gel and exist
Detecting under ultraviolet gel imaging instrument, detection wavelength is 302nm.Take pictures (Figure 12).
Testing result illustrates:
WspR albumen (pseudomonas aeruginosa abduction delivering) applied sample amount 10ul, through SDS-PAGE gel electrophoresis, with albumen
Marker comparison, this molecular weight of albumen, at about 40KD, is consistent with actual molecular weight；Protein band fluorescence intensity is very strong simultaneously,
Illustrate that this protein induced expression is the highest.Figure 12 explanation.
Claims (4)
1. fluorescence probe chloromethyl diazole rhodamine compound R H-MCl
。
2. the preparation method of fluorescence probe chloromethyl diazole rhodamine compound described in claim 1, it is characterised in that by such as
Under step carry out:
The synthesis of fluorescence probe:
(1) preparation of rhodamine hydrazides: take absolute ethyl alcohol and be placed in flask, rhodamine B 10.4mmol is dissolved in ethanol, so
After 80% hydrazine hydrate 133mmol is slowly added dropwise at ambient temperature in mixed system, after dropping, mixture is heated back
Stream, TLC monitors, and reacts the complete room temperature that is cooled to, and decompression removes partial solvent, pours in frozen water, has precipitation to produce, suction filtration,
To dark yellow solid；
(2) preparation of chloromethyl diazole rhodamine: monoxone 1.1mmol and POCl3 5 ml is mixed, is heated to reflux
0.5-1 hour, afterwards, being dividedly in some parts rhodamine B hydrazides 1.1mmol, TLC monitoring, question response is cooled to after completing, and adds few
Amount dichloromethane to room temperature and is poured in mixture of ice and water, and regulation pH is 8, with dichloromethane extraction crude product to inorganic layer solution
Till when color is deeper than organic layer solution color, merge organic layer, wash with 5ml × 2 saturated aqueous common salt, separate organic phase, use
Anhydrous magnesium sulfate is dried and stands overnight, and leaches drier, and vacuum rotary steam removes excess of solvent, and gained crude product dichloromethane/
Volume ratio 20:1 of methyl alcohol, pillar layer separation, obtain target compound RH-MCl.
3. chloromethyl diazole rhodamine compound application in terms of protein fluorescence probe mark described in claim 1.
4. the application described in claim 3, the method that wherein protein fluorescence is marked by fluorescence probe is as follows:
Protein s DS-PAGE gel electrophoresis labeling method:
1. weigh 0.055gRH-MCl to be dissolved in the acetum that 100ml volume ratio is 2%, be configured to the spy that concentration is 1000uM
Pin mother liquor, is diluted to concentration by this mother liquor with the acetum of 2% and is respectively the probe solution of 5uM, 10uM, 20uM, 40uM；
The most conventionally carrying out SDS-PAGE protein example gel electrophoresis, protein example uses commercially available Premixed
Protein Marker (Low) standard items；
3. take 4 pieces fixing after protein example gel be respectively placed in fluorescence probe concentration be 5uM, 10 uM, 20 uM, 40uM
Solution marks 20min, 80 rpm decolorization swinging tables are washed with deionized 2hr, take out gel at ultraviolet gel imaging instrument
Lower detection, detection wavelength is 302nm；
Analyze through software Gel-pro Analyzer and draw optimal probe label concentration；
The most separately take 4 pieces fixing after protein example gel be placed in the solution that fluorescence probe concentration is 20uM, mark respectively
5min, 10 min, 20 min, 40min, be washed with deionized 2hr on 80 rpm decolorization swinging tables, takes out gel and coagulates in ultraviolet
Detecting under glue imager, detection wavelength is 302nm；
Analyze through software Gel-pro Analyzer and draw the probe optimum mark time；
The most separately take 4 pieces fixing after protein example gel be placed in the solution that probe is 20uM, after mark 20min, respectively
After 80 rpm decolorization swinging tables are washed with deionized 1hr, 2hr, 3hr, 4hr, take out gel under ultraviolet gel imaging instrument
Detection, detection wavelength is 302nm, analyzes through software Gel-pro Analyzer and draws the optimal wash time of probe.
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| 高勇，等: "罗丹明荧光探针在生化分析中的应用", 《化学通报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112390763A (en) * | 2019-08-15 | 2021-02-23 | 上海交通大学 | Photosensitive compound, preparation method and application thereof, and photosensitive protein fixing gel containing photosensitive compound |
| CN112390763B (en) * | 2019-08-15 | 2024-02-27 | 水熊健康科技(南通)有限公司 | Photosensitive compound, preparation method and application thereof and photosensitive protein immobilized gel containing photosensitive compound |
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| Publication number | Publication date |
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| CN105859705B (en) | 2019-02-26 |
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