CN104607143A - Iron and nickel magnetic microsphere, preparation method thereof and application thereof in protein purification - Google Patents
Iron and nickel magnetic microsphere, preparation method thereof and application thereof in protein purification Download PDFInfo
- Publication number
- CN104607143A CN104607143A CN201510064372.5A CN201510064372A CN104607143A CN 104607143 A CN104607143 A CN 104607143A CN 201510064372 A CN201510064372 A CN 201510064372A CN 104607143 A CN104607143 A CN 104607143A
- Authority
- CN
- China
- Prior art keywords
- iron
- nickel
- magnetic
- preparation
- magnetic microsphere
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Soft Magnetic Materials (AREA)
Abstract
The invention provides an iron and nickel magnetic microsphere, a preparation method thereof and application thereof in protein purification, and relates to the field of magnetic microspheres. The preparation method comprises the steps of putting soluble molysite and soluble nickel salt into ethylene glycol, adding natrium aceticum, stirring for 30 minutes at 80 DEG C, then adding cholamine, reacting under the nitrogen protection at 180-210 DEG C for 6 hours, cooling to room temperature, and washing fully to obtain the iron and nickel magnetic microsphere. The synthesized iron and nickel microsphere can be directly used for purifying target proteins with histidine labels. The magnetic microsphere provided by the invention is convenient to separate, is relatively stable in structure and property, and is low in cost; moreover, the preparation method provided by the invention is simple, and the large-scale industrial production is easy; the invention provides a relatively rapid and easy-to-use method for purifying the proteins with histidine labels.
Description
Technical field
The present invention relates to magnetic microsphere, particularly relate to a kind of iron-nickel magnetic microballoon, its preparation method and the application of this magnetic microsphere in protein purification.
Background technology
Magnetic macromolecular microsphere is a kind of novel magnetic materials that developed recently gets up, and is magnetic inorganic particle to be combined by proper method the complex microsphere with certain magnetic and special construction formed with organic polymer.Numerous characteristics that magnetic composite microsphere not only has common polymer microsphere also have magnetic responsiveness, so can not only give its surface functional group by the method such as copolymerization and surface modification, can also have guide function under additional magnetic fields.At present, magnetic composite microsphere has been widely used in the numerous areas such as biomedicine, cytology and separation engineering.
The particle diameter of magnetic microsphere is little, specific area is large, surface is containing active group, therefore coupling capacity is large, magnetic microsphere covalent bond by the aglucon of target protein identification and Reversible binding, then, can directly be put into the mixed solution containing target protein by it, after target protein and magnetic microsphere are combined closely, external magnetic field is utilized to be separated.Whole separation process does not need the pH value to mixed solution, temperature, ionic strength and dielectric constant to adjust, thus avoids the loss of protein in traditional separation process.Generally speaking, with the protein in Beads enrichment cell pyrolysis liquid, need hardly to anticipate, compared with additive method, non-specific binding is also less.
The histidine-tagged production process being widely used in protein, utilize coordination in conjunction with nickel ion Ago-Gel can fast and highly selective absorption with histidine-tagged target protein, improve the separation and purification speed of protein, reduce protein purification cost, several functions protein has adopted this production technology to complete merchandized handling and application.In the production process of these functional proteins, coordination is high in conjunction with the Ago-Gel price of nickel ion, in separation and purification process, shared production cost proportion is maximum, therefore, and the bottleneck that exploitation is fast, efficient, protein separation method that is low cost is gpa protein development always.
Summary of the invention
For solving the problem, the invention provides a kind of iron-nickel magnetic microballoon, its preparation method and the application in protein purification.
Inventive concept of the present invention is: coordination is the Separation of Proteins material of extensive use in protein production in conjunction with the Ago-Gel (Ni-NTAagarose) of nickel ion, and its separation principle is the nickel ion (Ni that can identify with histidine-tagged protein on gel
2+), and be combined on gel by nickel ion absorption, by suitable buffer solution by foreign protein wash-out, and with histidine-tagged protein bound on gel, reach the object of separation and purification.Coordination is very high in conjunction with Ago-Gel (Ni-NTA agarose) price of nickel ion, therefore inventor is being devoted to how to obtain more cheap Separation of Proteins material always.
Inventor, through large quantifier elimination, has obtained a kind of iron-nickel magnetic microballoon of cheapness finally, and is directly used in the purifying of band histidine-tagged protein, and the present invention is achieved in that a kind of preparation method of iron-nickel magnetic microballoon, comprises the following steps:
A. soluble ferric iron salt, soluble nickel salt are placed in ethylene glycol, add sodium acetate, at 80 DEG C, in oil bath pan, stir 30min;
B. monoethanolamine is added, N
2in 180-210 DEG C of stirring reaction 6h under protection, after reaction terminates, stop logical N
2, naturally cool to 20-25 DEG C;
C. the product obtained in step b is spent deionized water 5-7 time, each washing 5 minutes, obtained hydration magnetic microsphere;
D. hydration magnetic microsphere is adopted successively percent by volume be 20%, 40%, 60% and 80% ethanol water respectively clean 2 times, use absolute ethanol washing again 5 times, washing afterproduct is put into open-top receptacle, drying at room temperature is volatilized completely to ethanol, vacuum drying 12h at 80 DEG C, obtains iron-nickel magnetic microballoon.
Preferably, described step a soluble ferric iron salt be in ferric nitrate, iron chloride, ferric sulfate or three's hydrate any one.
Preferably, described step a soluble nickel salt be in nickel nitrate, nickel chloride, nickelous sulfate or three's hydrate any one.
Preferably, the mol ratio of raw material is soluble ferric iron salt: soluble nickel salt: sodium acetate: ethylene glycol: monoethanolamine=1:0.5 ~ 2:1.3 ~ 2.6:100 ~ 250:80 ~ 110.
Further, the mol ratio of raw material is soluble ferric iron salt: soluble nickel salt: sodium acetate: ethylene glycol: monoethanolamine=1:1:1.46:235:100.
The present invention is the iron-nickel magnetic microballoon prepared of claimed said method itself also, and this iron-nickel magnetic microballoon is NiFe
2o
4.
Another object of the present invention is the application of the above-mentioned iron-nickel magnetic microballoon of protection in protein purification, without the need to additionally modifying the separation and purification that can directly apply to the histidine-tagged target protein of band after the preparation of iron-nickel magnetic microballoon.
Test through inventor confirms, magnetic microsphere provided by the invention has strong binding ability to the histidine-tagged albumen of band.Magnetic microsphere is mixed with crude protein extract, by centrifugal or magnet adsorption, isolate the magnetic microsphere with target protein, by the magnetic microsphere with target protein with after the cleaning of binding buffer liquid, then elute with histidine-tagged target protein from magnetic microsphere with the elution buffer containing high concentration imidazoles.
The histidine-tagged principle for protein purification is: histidine is the amino acid with heterocycle, and each histidine contains an imidazole group, and imidazole group is combined with some metal ion (particularly nickel ion) coordination by lone pair electrons.Histidine-tagged is 6 continuous print His segments on protein, has the affinity of height can be used for the affinity purification of protein with nickel ion.
Primary object of the present invention is: prepare nickeliferous magnetic microsphere according to thermal decomposition method, and magnetic microsphere is modified without other and directly relied on the absorption of the nickel ion of magnetic microsphere itself to reach separation and purification object with histidine-tagged target protein.Magnetic microsphere purifying histidine-tagged protein combines the advantage of immobilization metal affinity chromatography and magnetic nano-particle, transition metal ions and amino acid residue can be utilized as the coordination of the imidazole radicals of histidine, optionally purifying has the protein of affinity to metal ion, Magnetic Isolation and the high-adsorption-capacity advantage of microballoon can be given full play to again, simple economy, rapidly and efficiently.
Compared with prior art, the invention has the beneficial effects as follows: (1). the iron-nickel magnetic microballoon synthesized by the present invention can be directly used in purifying with histidine-tagged target protein, modify without the need to carrying out other on magnetic ball surface, as antibody modification, the nickel ion of magnetic microsphere itself is relied on to be directly used in protein purification.(2). the microballoon that the present invention obtains is magnetic, good dispersion, and effect is rapidly and efficiently, is convenient to be separated, and its structures and characteristics is more stable, preserves and still have good magnetic in 2 years in the aqueous solution.(3). preparation method of the present invention is simple, cost is low, easily carry out large-scale industrial production.
Accompanying drawing explanation
The transmission electron microscope photo of Fig. 1 magnetic microsphere;
Fig. 2 is the dispersed testing result figure of the embodiment of the present invention 1 magnetic microsphere, wherein: the iron-nickel magnetic microballoon 1, after dispersion, and 2, iron-nickel magnetic microballoon under external magnetic field;
Fig. 3 is the embodiment of the present invention 1 magnetic microsphere XRD collection of illustrative plates;
Fig. 4 is the colibacillary fluorescence microscope testing result expressing the histidine-tagged green fluorescent protein of band;
Fig. 5 is that the magnetic microsphere of the quality such as application examples and the coordination Ago-Gel in conjunction with nickel ion is in conjunction with the comparison diagram with histidine-tagged green fluorescent protein in supernatant after bacteria lysis, wherein, 1, protein molecular weight standard, 2, coordination in conjunction with the Ago-Gel of nickel ion, 3, magnetic microsphere;
Fig. 6 is application examples magnetic microsphere and the Ago-Gel separating effect comparison diagram of coordination in conjunction with nickel ion, wherein, and 1,0.5g magnetic microsphere, 2,0.1g coordination is in conjunction with the Ago-Gel of nickel ion.
Detailed description of the invention
Below by detailed description of the invention, the present invention will be further described, but be not limited to embodiment.
If no special instructions, the present invention is raw materials used all commercially, and as preferably, raw material involved in the present invention is as follows:
NiSO
46H
2o (Chemical Reagent Co., Ltd., Sinopharm Group), FeCl
36H
2o (Chemical Reagent Co., Ltd., Sinopharm Group), sodium acetate (Shenyang chemical reagent Co., Ltd), ethylene glycol (Chemical Reagent Co., Ltd., Sinopharm Group), monoethanolamine (Chemical Reagent Co., Ltd., Sinopharm Group).
The concrete model of experimental facilities involved in the present invention and manufacturer as follows:
Magnetic stirring apparatus (DF-101S type, Ying Yu Yu Hua instrument plant of Gongyi City), vacuum drying chamber (DZF-6020 type, Shanghai Yiheng Scientific Instruments Co., Ltd).
Embodiment 1
The drug dosage of table 1 embodiment 1 and output
Utilize decomposition method for preparing Ni Fe
2o
4magnetic microsphere:
A. by FeCl
36H
2o, NiSO
46H
2o is placed in ethylene glycol, adds sodium acetate, at 80 DEG C in oil bath pan, stirs 30min;
B. monoethanolamine is added, N
2in 180 DEG C of stirring reaction 6h under protection, after reaction terminates, stop logical N
2, naturally cool to 25 DEG C;
C. the product obtained in step b is spent deionized water 5-7 time, each washing 5 minutes, obtained hydration magnetic microsphere;
D. hydration magnetic microsphere is adopted successively percent by volume be 20%, 40%, 60% and 80% ethanol water each 100 milliliters washing 2 times, again with absolute ethyl alcohol 100 milliliters washing 5 times, washing afterproduct is put into open-top receptacle, drying at room temperature is volatilized completely to ethanol, vacuum drying 12h at 80 DEG C, obtain iron-nickel magnetic microballoon, gained magnetic microsphere powder quality is 0.71g.
Magnetic microsphere dispersiveness experiment: suspended in deionized water by magnetic microsphere, detects dispersed, leans against on test tube outer wall detect magnetic microsphere and magnetic fields feature with magnet.The microballoon dispersiveness be illustrated in figure 2 by the method for embodiment 1 is obtained characterizes: (1) is dispersed in aqueous phase preferably for iron-nickel magnetic microballoon, (2) be under the existent condition of external magnetic field, iron-nickel magnetic microballoon is rapidly to external magnetic field enrichment, and the aqueous solution becomes clarification.This tests proof: obtained magnetic microsphere favorable dispersibility, and can enrichment in magnetic field.Utilize x-ray powder diffraction instrument to detect magnetic microsphere powder, XRD testing result as shown in Figure 3: by the comparison of XRD testing result and standard spectrum chart database, determine that crystallite is square, crystal formation is NiFe
2o
4, utilize Scherrer formulae discovery to go out particle diameter for 6.75nm.
Embodiment 2
The drug dosage of table 2 embodiment 2 and output
A. by FeCl
36H
2o, NiSO
46H
2o is placed in ethylene glycol, adds sodium acetate, at 80 DEG C in oil bath pan, stirs 30min;
B. monoethanolamine is added, N
2in 200 DEG C of stirring reaction 6h under protection, after reaction terminates, stop logical N
2,naturally cool to 25 DEG C;
C. the product obtained in step b is spent deionized water 5-7 time, each washing 5 minutes, obtained hydration magnetic microsphere;
D. hydration magnetic microsphere is adopted successively percent by volume be 20%, 40%, 60% and 80% ethanol water each 100 milliliters washing 2 times, again with absolute ethyl alcohol 100 milliliters washing 5 times, washing afterproduct is put into open-top receptacle, drying at room temperature is volatilized completely to ethanol, vacuum drying 12h at 80 DEG C, obtain iron-nickel magnetic microballoon, gained magnetic microsphere powder quality is 0.68g.
Embodiment 3
The drug dosage of table 3 embodiment 3 and output
A. by Fe (NO
3)
39H
2o, NiCl
26H
2o is placed in ethylene glycol, adds sodium acetate, at 80 DEG C in oil bath pan, stirs 30min;
B. monoethanolamine is added, N
2in 210 DEG C of stirring reaction 6h under protection, after reaction terminates, stop logical N
2, naturally cool to 22 DEG C;
C. the product obtained in step b is spent deionized water 5-7 time, each washing 5 minutes, obtained hydration magnetic microsphere;
D. hydration magnetic microsphere is adopted successively percent by volume be 20%, 40%, 60% and 80% ethanol water each 100 milliliters washing 2 times, again with absolute ethyl alcohol 100 milliliters washing 5 times, washing afterproduct is put into open-top receptacle, drying at room temperature is volatilized completely to ethanol, vacuum drying 12h at 80 DEG C, obtain iron-nickel magnetic microballoon, gained magnetic microsphere powder quality is 1.23g.
Embodiment 4
The drug dosage of table 4 embodiment 4 and output
A. by Fe
2(SO
4)
3, NiCl
2be placed in ethylene glycol, add sodium acetate, at 80 DEG C in oil bath pan, stir 30min;
B. monoethanolamine is added, N
2in 180 DEG C of stirring reaction 6h under protection, after reaction terminates, stop logical N
2, naturally cool to 25 DEG C;
C. the product obtained in step b is spent deionized water 5-7 time, each washing 5 minutes, obtained hydration magnetic microsphere;
D. hydration magnetic microsphere is adopted successively percent by volume be 20%, 40%, 60% and 80% ethanol water each 100 milliliters washing 2 times, again with absolute ethyl alcohol 100 milliliters washing 5 times, washing afterproduct is put into open-top receptacle, drying at room temperature is volatilized completely to ethanol, vacuum drying 12h at 80 DEG C, obtain iron-nickel magnetic microballoon, gained magnetic microsphere powder quality is 0.44g.
Embodiment 5
The drug dosage of table 5 embodiment 5 and output
A. by Fe
2(SO
4)
39H
2o, Ni (NO
3)
26H
2o is placed in ethylene glycol, adds sodium acetate, at 80 DEG C in oil bath pan, stirs 30min;
B. monoethanolamine is added, N
2in 180 DEG C of stirring reaction 6h under protection, after reaction terminates, stop logical N
2, naturally cool to 25 DEG C;
C. the product obtained in step b is spent deionized water 5-7 time, each washing 5 minutes, obtained hydration magnetic microsphere;
D. hydration magnetic microsphere is adopted successively percent by volume be 20%, 40%, 60% and 80% ethanol water each 100 milliliters washing 2 times, again with absolute ethyl alcohol 100 milliliters washing 5 times, washing afterproduct is put into open-top receptacle, drying at room temperature is volatilized completely to ethanol, vacuum drying 12h at 80 DEG C, obtain iron-nickel magnetic microballoon, gained magnetic microsphere powder quality is 1.14g.
Embodiment 6
The drug dosage of table 6 embodiment 6 and output
A. by Fe (NO
3)
3, NiSO
4be placed in ethylene glycol, add sodium acetate, at 80 DEG C in oil bath pan, stir 30min;
B. monoethanolamine is added, N
2in 180 DEG C of stirring reaction 6h under protection, after reaction terminates, stop logical N
2, naturally cool to 20 DEG C;
C. the product obtained in step b is spent deionized water 5-7 time, each washing 5 minutes, obtained hydration magnetic microsphere;
D. hydration magnetic microsphere is adopted successively percent by volume be 20%, 40%, 60% and 80% ethanol water each 100 milliliters washing 2 times, again with absolute ethyl alcohol 100 milliliters washing 5 times, washing afterproduct is put into open-top receptacle, drying at room temperature is volatilized completely to ethanol, vacuum drying 12h at 80 DEG C, obtain iron-nickel magnetic microballoon, gained magnetic microsphere powder quality is 0.71g.
Embodiment 7
The drug dosage of table 7 embodiment 7 and output
A. by FeCl
3, Ni (NO
3)
2be placed in ethylene glycol, add sodium acetate, at 80 DEG C in oil bath pan, stir 30min;
B. monoethanolamine is added, N
2in 180 DEG C of stirring reaction 6h under protection, after reaction terminates, stop logical N
2, naturally cool to 25 DEG C;
C. the product obtained in step b is spent deionized water 5-7 time, each washing 5 minutes, obtained hydration magnetic microsphere;
D. hydration magnetic microsphere is adopted successively percent by volume be 20%, 40%, 60% and 80% ethanol water each 100 milliliters washing 2 times, again with absolute ethyl alcohol 100 milliliters washing 5 times, washing afterproduct is put into open-top receptacle, drying at room temperature is volatilized completely to ethanol, vacuum drying 12h at 80 DEG C, obtain iron-nickel magnetic microballoon, gained magnetic microsphere powder quality is 0.69g.
Application examples
Solution involved in application examples and medicine specific as follows:
1, PBS buffer solution
Weigh 4g NaCl, 0.1g KCl, 0.71g Na
2hPO
4, 0.135g KH
2pO
4, add 400mL deionized water dissolving, drip concentrated hydrochloric acid and adjust pH7.4, deionized water is settled to 500mL, room temperature preservation after autoclave sterilization.
2, IPTG solution
Weigh 0.24g IPTG, 40mL sterilized water is settled to 50mL after dissolving, and 0.22 μm of membrane filtration is degerming, the packing of 1mL/ part ,-20 DEG C of preservations.
3, binding buffer liquid:
Weigh 0.68g imidazoles, the PBS solution sterilized with 0.5L is dissolved.
4, elution buffer:
Weigh 17g imidazoles, be dissolved in the sterilized PBS solution of 0.5L.
5, coordination is in conjunction with the Ago-Gel of nickel ion
Ni-NTAagarose is QIAGEN Products.
First should be placed in expression in escherichia coli by with histidine-tagged green fluorescent protein by use-case, this operation can adopt the conventional method of this area to carry out, and the present invention is not particularly limited this.
Application examples magnetic microsphere and coordination are in conjunction with the control experiment of the histidine-tagged green fluorescent protein of the agarose gel purification band of nickel ion:
A, by can express with the Escherichia coli of histidine-tagged green fluorescent protein be inoculated in LB fluid nutrient medium (containing antibiotic) 37 DEG C cultivate 12h, add IPTG in 25 DEG C of Fiber differentiation 12h.
Utilize fluorescence microscope whether to detect green fluorescent protein at expression in escherichia coli, as shown in Figure 4, can see that thalline sends green fluorescence clearly, prove green fluorescent protein at expression in escherichia coli.
Collected by centrifugation thalline, with the resuspended rear ultrasonication thalline of PBS buffer solution and centrifugal acquisition supernatant, is used for purified green fluorescent protein by supernatant;
B. washed 2-3 time with PBS buffer solution, binding buffer liquid respectively in conjunction with the Ago-Gel of nickel ion with the coordination of purchase by the magnetic microsphere of preparation in embodiment 1, centrifugal rear removal buffer solution, weigh, load in test tube, mark is good for subsequent use;
C. be equipped with in step b in the magnetic microsphere after cleaning and two test tubes of coordination in conjunction with the Ago-Gel of nickel ion and add the supernatant obtained in 500 μ L step a respectively, abundant mixing, by coordination in conjunction with the Ago-Gel test tube of nickel ion in the centrifugal 6min of 8000rpm; Magnetic microsphere test tube then uses magnet near the side of test tube, magnetic microsphere under the existent condition of external magnetic field, rapid enrichment, solution become clarification, magnetic microsphere is separated;
D. remove coordination in conjunction with the foreign protein on the Ago-Gel of nickel ion or magnetic microsphere with the cleaning of binding buffer liquid, after washing 3 times, add elution buffer and wash down with histidine-tagged green fluorescent protein, collect eluent respectively and preserve in 4 DEG C of refrigerators;
E. get eluent 80 μ L in steps d, add 20 μ L, 5 times of sample-loading buffers (LoadingBuffer), mixing, in 95 DEG C of heating 10min, then in the centrifugal 10min of 10000rpm, get supernatant and carry out SDS-PAGE electrophoresis detection.
By etc. quality coordination in conjunction with the Ago-Gel (2) of nickel ion and magnetic microsphere (3) respectively after lysis gained supernatant be combined, after the washing of binding buffer liquid, utilize SDS-PAGE method to detect, the results are shown in Figure 5.Magnetic microsphere can in conjunction with histidine-tagged green fluorescent protein as can be seen from Figure 5, but binding ability is more weak in conjunction with the Ago-Gel of nickel ion than coordination.Obtaining its molecular weight as calculated with histidine-tagged green fluorescent protein is 26.9kD, and the molecular weight corresponding marker (protein molecular weight standard) is respectively 20.1kD and 29.0kD.
Fig. 6 is application examples magnetic microsphere (1) and Ago-Gel (2) the separating effect comparison diagram of coordination in conjunction with nickel ion, from Fig. 6, result is known, 0.5 gram of magnetic microsphere is suitable in conjunction with the Ago-Gel separation and purification ability of nickel ion with 0.1 gram of coordination, and the magnetic microsphere that the present invention can be adopted to prepare carries out purifying in conjunction with the Ago-Gel of nickel ion to band histidine-tagged protein matter to replace coordination.
From above-mentioned application examples, the present invention is that the purifying of band histidine-tagged protein matter provides a relatively quick and wieldy method, synthesized iron-nickel magnetic microballoon can be used in purifying with histidine-tagged green fluorescent protein, the magnetic microsphere that the present invention obtains is convenient to be separated, structures and characteristics is more stable, and preparation method of the present invention is simple, easy to operate, cost is low, easily carries out large-scale industrial production.
Claims (8)
1. a preparation method for iron-nickel magnetic microballoon, is characterized in that, comprises the following steps:
A. soluble ferric iron salt, soluble nickel salt are placed in ethylene glycol, add sodium acetate, at 80 DEG C in oil bath pan, stir 30min;
B. monoethanolamine is added, N
2in 180-210 DEG C of stirring reaction 6h under protection, after reaction terminates, stop logical N
2, naturally cool to 20-25 DEG C;
C. the product obtained in step b is spent deionized water 5-7 time, each washing 5 minutes, obtained hydration magnetic microsphere;
D. hydration magnetic microsphere is adopted successively percent by volume be 20%, 40%, 60% and 80% ethanol water respectively clean 2 times, use absolute ethanol washing again 5 times, washing afterproduct is put into open-top receptacle, drying at room temperature is volatilized completely to ethanol, vacuum drying 12h at 80 DEG C, obtains iron-nickel magnetic microballoon.
2. the preparation method of a kind of iron-nickel magnetic microballoon according to claim 1, is characterized in that, described step a soluble ferric iron salt be in ferric nitrate, iron chloride, ferric sulfate or three's hydrate any one.
3. the preparation method of a kind of iron-nickel magnetic microballoon according to claim 1, is characterized in that, described step a soluble nickel salt be in nickel nitrate, nickel chloride, nickelous sulfate or three's hydrate any one.
4. the preparation method of a kind of iron-nickel magnetic microballoon according to claim 1, it is characterized in that, the mol ratio of raw material is soluble ferric iron salt: soluble nickel salt: sodium acetate: ethylene glycol: monoethanolamine=1:0.5 ~ 2:1.3 ~ 2.6:100 ~ 250:80 ~ 110.
5. the preparation method of a kind of iron-nickel magnetic microballoon according to claim 4, is characterized in that, the mol ratio of raw material is soluble ferric iron salt: soluble nickel salt: sodium acetate: ethylene glycol: monoethanolamine=1:1:1.46:235:100.
6. an iron-nickel magnetic microballoon obtains according to method preparation described in claim 1, and it is characterized in that, iron-nickel magnetic microballoon is NiFe
2o
4.
7. by the application of iron-nickel magnetic microballoon in protein purification that method according to claim 1 obtains, it is characterized in that, without the need to additionally modifying the separation and purification that can directly apply to the histidine-tagged target protein of band after the preparation of iron-nickel magnetic microballoon.
8. the application of iron-nickel magnetic microballoon according to claim 7 in protein purification, it is characterized in that, comprise the following steps: magnetic microsphere is mixed with crude protein extract, by centrifugal or magnet adsorption, isolate the magnetic microsphere with target protein, by the magnetic microsphere with target protein with after the cleaning of binding buffer liquid, then elute with histidine-tagged target protein from magnetic microsphere with the elution buffer containing high concentration imidazoles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510064372.5A CN104607143A (en) | 2015-02-06 | 2015-02-06 | Iron and nickel magnetic microsphere, preparation method thereof and application thereof in protein purification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510064372.5A CN104607143A (en) | 2015-02-06 | 2015-02-06 | Iron and nickel magnetic microsphere, preparation method thereof and application thereof in protein purification |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104607143A true CN104607143A (en) | 2015-05-13 |
Family
ID=53141947
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510064372.5A Pending CN104607143A (en) | 2015-02-06 | 2015-02-06 | Iron and nickel magnetic microsphere, preparation method thereof and application thereof in protein purification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104607143A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107175089A (en) * | 2017-06-06 | 2017-09-19 | 大连民族大学 | A kind of iron-nickel magnetic microballoon of cobalt ions that adulterates and its application |
| CN109453739A (en) * | 2018-10-15 | 2019-03-12 | 南京农业大学 | Ni/Fe3O4@C composite and its preparation method and application |
| CN109569546A (en) * | 2018-07-05 | 2019-04-05 | 华侨大学 | A kind of preparation method and application of the chelated mineral magnetic nanoparticle for histidine-tagged protein purifying |
| CN114524470A (en) * | 2022-02-24 | 2022-05-24 | 安徽工程大学 | Nickel ferrite nano particle and green synthesis method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102500378A (en) * | 2011-11-03 | 2012-06-20 | 东华大学 | Preparation method for magnetically separable photo-catalyst nanospheres of egg-like structure |
| US20120264188A1 (en) * | 2011-04-15 | 2012-10-18 | Korea Institute Of Ceramic Engineering And Technology | Method for preparing ni-containing magnetic mesoporous silica whose surface strongly binds histidine-tagged protein, protein-binding material for degrading toxic aromatic compound comprising the magnetic mesoporous silica, and method for degrading toxic aromatic compound using the magnetic mesoporous silica |
| CN103665278A (en) * | 2012-09-16 | 2014-03-26 | 上海遥科生物科技发展有限公司 | Core-shell type magnetic composite microsphere for separation and purification of recombinant proteins and preparation method thereof |
| CN103897123A (en) * | 2014-03-13 | 2014-07-02 | 复旦大学 | Method for preparing nuclear-shell type magnetic composite microsphere with surface rich in nickel ions and application of nuclear-shell type magnetic composite microsphere |
-
2015
- 2015-02-06 CN CN201510064372.5A patent/CN104607143A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120264188A1 (en) * | 2011-04-15 | 2012-10-18 | Korea Institute Of Ceramic Engineering And Technology | Method for preparing ni-containing magnetic mesoporous silica whose surface strongly binds histidine-tagged protein, protein-binding material for degrading toxic aromatic compound comprising the magnetic mesoporous silica, and method for degrading toxic aromatic compound using the magnetic mesoporous silica |
| CN102500378A (en) * | 2011-11-03 | 2012-06-20 | 东华大学 | Preparation method for magnetically separable photo-catalyst nanospheres of egg-like structure |
| CN103665278A (en) * | 2012-09-16 | 2014-03-26 | 上海遥科生物科技发展有限公司 | Core-shell type magnetic composite microsphere for separation and purification of recombinant proteins and preparation method thereof |
| CN103897123A (en) * | 2014-03-13 | 2014-07-02 | 复旦大学 | Method for preparing nuclear-shell type magnetic composite microsphere with surface rich in nickel ions and application of nuclear-shell type magnetic composite microsphere |
Non-Patent Citations (2)
| Title |
|---|
| JINYOUNG CHUN ET AL.: ""Easy access to efficient magnetically recyclable separation of histidine-tagged proteins using superparamagnetic nickel ferrite nanoparticle clusters"", 《JOURNAL OF MATERIALS CHEMISTRY》 * |
| SASMITA MOHAPATRA ET AL.: ""One-pot synthesis of uniform and spherically assembled functionalized MFe2O4 (M=Co，Mn，Ni) nanoparticles"", 《COLLOIDS AND SURFACES A:PHYSICOCHEMICAL AND ENGINEERING ASPECTS》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107175089A (en) * | 2017-06-06 | 2017-09-19 | 大连民族大学 | A kind of iron-nickel magnetic microballoon of cobalt ions that adulterates and its application |
| CN108043372A (en) * | 2017-06-06 | 2018-05-18 | 大连民族大学 | A kind of iron-nickel magnetic microballoon for adulterating cobalt ions |
| CN109569546A (en) * | 2018-07-05 | 2019-04-05 | 华侨大学 | A kind of preparation method and application of the chelated mineral magnetic nanoparticle for histidine-tagged protein purifying |
| CN109569546B (en) * | 2018-07-05 | 2021-05-18 | 华侨大学 | Preparation method and application of chelated metal magnetic nanoparticles for purifying histidine-tagged protein |
| CN109453739A (en) * | 2018-10-15 | 2019-03-12 | 南京农业大学 | Ni/Fe3O4@C composite and its preparation method and application |
| CN114524470A (en) * | 2022-02-24 | 2022-05-24 | 安徽工程大学 | Nickel ferrite nano particle and green synthesis method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Metal–organic framework UiO-66 modified magnetite@ silica core–shell magnetic microspheres for magnetic solid-phase extraction of domoic acid from shellfish samples | |
| EP2709950B1 (en) | Chemically modified graphene | |
| Okoli et al. | Application of magnetic iron oxide nanoparticles prepared from microemulsions for protein purification | |
| CN103212377B (en) | Preparation method of agarose immune magnetic microspheres and applications thereof | |
| CN104607143A (en) | Iron and nickel magnetic microsphere, preparation method thereof and application thereof in protein purification | |
| Wang et al. | Selective separation of bovine hemoglobin using magnetic mesoporous rare-earth silicate microspheres | |
| Han et al. | Fabrication of a core-shell-shell magnetic polymeric microsphere with excellent performance for separation and purification of bromelain | |
| Atta et al. | Removal of thorium from water using modified magnetite nanoparticles capped with rosin amidoxime | |
| Zou et al. | Ferroferric oxide/L-cysteine magnetic nanospheres for capturing histidine-tagged proteins | |
| Ni et al. | Preparation of core–shell structure Fe3O4@ SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for his‐tag protein purification | |
| He et al. | Facial synthesis of nickel (II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins | |
| Zhang et al. | Dual-emitting metal–organic frameworks for ratiometric fluorescence detection of fluoride and Al3+ in sequence | |
| CN108043372A (en) | A kind of iron-nickel magnetic microballoon for adulterating cobalt ions | |
| CN106366195B (en) | PD-L1 antibody immunomagnetic beads and preparation method thereof | |
| Cao et al. | Preferential adsorption of flavonoids from peanut shell by amino‐modified Fe3O4 nanoparticles (MNP‐NH2) | |
| Feng et al. | Magnetic technologies and green solvents in extraction and separation of bioactive molecules together with biochemical objects: current opportunities and challenges | |
| CN105396558A (en) | Affinity adsorption material based on anti-AFB1 nanometer antibody | |
| Park et al. | Functionalized Ni@ SiO 2 core/shell magnetic nanoparticles as a chemosensor and adsorbent for Cu2+ ion in drinking water and human blood | |
| Shi et al. | Preparation of IgY oriented conjugated Fe3O4 MNPs as immunomagnetic nanoprobe for increasing enrichment efficiency of Staphylococcus aureus based on adjusting the pH of the solution system | |
| Zhang et al. | Extraction of genomic DNA via superparamagnetic Fe3O4 magnetic colloidal nanocrystal clusters | |
| Wang et al. | Amino acid-based ionic liquid surface modification of magnetic nanoparticles for the magnetic solid-phase extraction of heme proteins | |
| CN106279421B (en) | CD133, CD24 and CD44 multiple antibody immunomagnetic beads and preparation method thereof | |
| Chen et al. | Purification of 6× His-tagged phycobiliprotein using zinc-decorated silica-coated magnetic nanoparticles | |
| KR101581576B1 (en) | Method for The Preparation of Amylose Magnetic Bead and Its Application for Protein Purification | |
| CN111057185B (en) | Functional magnetic material for selectively separating pepsin and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150513 |