CN105968207A - Novel method for immobilizing protein - Google Patents
Novel method for immobilizing protein Download PDFInfo
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- CN105968207A CN105968207A CN201610310137.6A CN201610310137A CN105968207A CN 105968207 A CN105968207 A CN 105968207A CN 201610310137 A CN201610310137 A CN 201610310137A CN 105968207 A CN105968207 A CN 105968207A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
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Abstract
The invention discloses a novel method for immobilizing protein, which adopts a sodium cyanoborohydride reduction method. The novel comprises the following steps: step 11: preparing epoxidized agarose; step 12: carrying out ammonification treatment on the epoxidized agarose obtained in the step 11; step 13: preparing a formyl carrier; step 14: coupling the protein with the formyl carrier. The method for immobilizing the protein, disclosed by the invention, does not need to be performed under an alkaline condition, has moderate conditions and has relatively small or no influence on natural properties of the protein.
Description
Technical field
The present invention relates to biomedical sector, specifically refer to the new method of a kind of protein immobilization.
Background technology
Immobilization of protein has many kind methods, it is most commonly used that at present and uses cyanogen bromide-activated agarose, then protein is received on the agarose of activation, this method needs to carry out in the basic conditions, busy meeting produces certain impact to conformation and the character of protein, thus the method is restricted on using.
Summary of the invention
It is an object of the invention to provide the new method of a kind of protein immobilization that need not and carry out in the basic conditions, the condition making protein immobilization is gentleer, reduces the impact on protein natural attribute.
The present invention is achieved through the following technical solutions: uses sodium cynoborohydride reducing process, comprises the following steps:
Step 11: prepare epoxidation agarose；
Step 12: the epoxidation agarose obtained in step 1 is carried out ammoniated treatment；
Step 13: prepare formyl carrier；
Step 14: protein and formyl carrier are carried out coupling.
Further, described step 11 is prepared epoxidation sepharose step and farther includes step 111:
Step 111: be suspended in cleaning the agarose after draining in redistilled water, it is mixed into sodium hydroxide that concentration is 2M successively and concentration is the epoxychloropropane of 5%, by this suspension in 40 DEG C of oscillating reactionss 2 hours, then proceed to sand core funnel, clean with substantial amounts of water sucking filtration；
Or step 111 is: the agarose blotted is washed on core and blots, it is mixed into two glycerin ethers the most respectively and sodium borohydride and concentration that concentration is 2mg/ml are the sodium hydroxide solution of 0.6M, this suspension is transferred to sand core funnel in 25 DEG C of vibration insulation reaction after 8 hours, washes glue with substantial amounts of water sucking filtration.
Further, in described step 14, the coupling step of protein and formyl carrier farther includes:
Step 141: protein and sodium cyanoborohydride are dissolved in the acetate buffer solution that concentration is 0.1M that PH is 6.4, also include the Alpha-Methyl mannoside of the manganese chloride of 1mM, the magnesium chloride of 1mM, the calcium chloride of 1mM and 0.2M in described acetate buffer solution；
Step 142: mixed with the formyl carrier prepared in step 13 by the solution obtained by step 141, puts 4 DEG C of agitation reactions overnight；
Step 143: be suspended from the glutaraldehyde solution of 1% by the gel obtained by step 142, also comprises the single-minded sugar of sodium cyanoborohydride and 0.2M, then puts 4 DEG C of agitation reactions overnight in described glutaraldehyde solution.
Step 144: it is in three (methylol) aminomethane buffer solution that 7.4 concentration are 1M that gel is placed in PH, also includes sodium cyanoborohydride, room temperature reaction 1 hour in described three (methylol) aminomethane buffer solution；
Step 145: after having reacted, is transferred to gel on sand core funnel, washes away unreacted sodium cyanoborohydride with redistilled water；
Step 146: wash gel with the acetate buffer solution that concentration is 0.2M that the sodium chloride of 2M, PH are 4.0 and the carbonic acid buffer that concentration is 0.1 that PH is 9.0 respectively, after being washed till neutrality with redistilled water, it is placed in three (methylol) aminomethane buffer solution that concentration is 0.2M that PH is 7.4, add preservative Sodium Azide, put in 4 DEG C of refrigerators and preserve.
Further, the acetate buffer solution also phosphoric acid buffer in described step 141.
The present invention has the following advantages and beneficial effect:
Need not method for immobilization of protein in the present invention carry out in the basic conditions, mild condition, the impact on protein natural attribute is less or does not affect.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment:
The present embodiment uses Con A Concanavalin, carries out as follows:
Step 11: prepare epoxidation agarose
Step 111: the agarose drained after cleaning 20 grams is suspended in the redistilled water of 30 milliliters, it is mixed into the 2M sodium hydroxide of 13 milliliters and the epoxychloropropane of 3 milliliters successively, the final concentration of each material is respectively as follows: 30% (v/v) agarose gel, 5% epoxychloropropane, 0.4M sodium hydroxide, by this suspension and 40 DEG C of oscillating reactionss 2 hours, then proceed to sand core funnel, clean with substantial amounts of water sucking filtration:；
Or step 111 is: 1 gram of agarose blotted is washed on core and blots, it is mixed into 1 milliliter of two glycerin ether and 1 milliliter of 0.6M sodium hydroxide solution containing 2 milligrams of every milliliter of sodium borohydrides the most respectively, proper to this suspension and 25 DEG C insulation reaction is transferred to sand core funnel after 8 hours, glue (in this reaction, temperature is the most important, too high or too low all affects reaction efficiency) is washed with substantial amounts of water sucking filtration.
Step 12: the epoxidation agarose obtained in step 1 is carried out ammoniated treatment
It is suspended from the strong aqua ammonia of 1.5 times of volumes after the agarose gel massive laundering of epoxy activation is clean, by this suspension 40 DEG C of incubated under agitation 1.5 hours, is proceeded to after react in sand core funnel, clean with redistilled water.
Step 13: prepare formyl carrier
In 1 gram of glutaraldehyde solution wanting to add 1 milliliter 25% in the ammonification agarose blotted and 82 milligrams of sodium cyanoborohydrides, this mixture is vibrated 2 hours at 40 DEG C of incubations, then cleans with redistilled water.
Step 14: protein and formyl carrier are carried out coupling
Step 141: the concentration that PH is 6.4 that the sodium cyanoborohydride of the Con A Concanavalin of 10 ~ 50 milligrams and 12 milligrams is dissolved in 1 milliliter is in 0.1M acetate buffer solution, also includes the Alpha-Methyl mannoside of the manganese chloride of 1mM, the magnesium chloride of 1mM, the calcium chloride of 1mM and 0.2M in described acetate buffer solution；
Step 142: by solution obtained in step 141 and 0.5 gram of formyl carrier mixing, put 4 DEG C of agitation reactions overnight；
Step 143: be suspended from the glutaraldehyde solution of 2 milliliter 1% by the gel obtained by step 142, also comprises the single-minded sugar of 48 milligrams of sodium cyanoborohydrides and 0.2M, then puts 4 DEG C of agitation reactions overnight in described glutaraldehyde solution.
Step 144: it is in three (methylol) aminomethane buffer solution that 7.4 concentration are 1M that gel is placed in 2 milliliters of PH, also includes 6.2 milligrams of sodium cyanoborohydrides, room temperature reaction 1 hour in described three (methylol) aminomethane buffer solution；
Step 145: after having reacted, is transferred to gel on sand core funnel, washes away unreacted sodium cyanoborohydride with redistilled water；
Step 146: wash gel with the acetate buffer solution that concentration is 0.2M that the sodium chloride of 2M, PH are 4.0 and the carbonic acid buffer that concentration is 0.1 that PH is 9.0 respectively, after being washed till neutrality with redistilled water, it is placed in three (methylol) aminomethane buffer solution that concentration is 0.2M that PH is 7.4, add preservative Sodium Azide, put in 4 DEG C of refrigerators and preserve.
The invention is not limited in aforesaid detailed description of the invention.The present invention expands to arbitrary new method or the step of process or any new combination disclosed in any this specification.
Claims (4)
1. the new method of a protein immobilization, it is characterised in that use sodium cynoborohydride reducing process, comprise the following steps:
Step 11: prepare epoxidation agarose；
Step 12: the epoxidation agarose obtained in step 1 is carried out ammoniated treatment；
Step 13: prepare formyl carrier；
Step 14: protein and formyl carrier are carried out coupling.
The new method of a kind of protein immobilization the most according to claim 1, it is characterised in that prepare epoxidation sepharose step in described step 11 and farther include step 111:
Step 111: be suspended in cleaning the agarose after draining in redistilled water, it is mixed into sodium hydroxide that concentration is 2M successively and concentration is the epoxychloropropane of 5%, by this suspension in 40 DEG C of oscillating reactionss 2 hours, then proceed to sand core funnel, clean with substantial amounts of water sucking filtration；
Or step 111 is: the agarose blotted is washed on core and blots, it is mixed into two glycerin ethers the most respectively and sodium borohydride and concentration that concentration is 2mg/ml are the sodium hydroxide solution of 0.6M, this suspension is transferred to sand core funnel in 25 DEG C of vibration insulation reaction after 8 hours, washes glue with substantial amounts of water sucking filtration.
The new method of a kind of protein immobilization the most according to claim 1, it is characterised in that in described step 14, the coupling step of protein and formyl carrier farther includes:
Step 141: protein and sodium cyanoborohydride are dissolved in the acetate buffer solution that concentration is 0.1M that PH is 6.4, also include the Alpha-Methyl mannoside of the manganese chloride of 1mM, the magnesium chloride of 1mM, the calcium chloride of 1mM and 0.2M in described acetate buffer solution；
Step 142: mixed with the formyl carrier prepared in step 13 by the solution obtained by step 141, puts 4 DEG C of agitation reactions overnight；
Step 143: be suspended from the glutaraldehyde solution of 1% by the gel obtained by step 142, also comprises the single-minded sugar of sodium cyanoborohydride and 0.2M, then puts 4 DEG C of agitation reactions overnight in described glutaraldehyde solution；
Step 144: it is in three (methylol) aminomethane buffer solution that 7.4 concentration are 1M that gel is placed in PH, also includes sodium cyanoborohydride, room temperature reaction 1 hour in described three (methylol) aminomethane buffer solution；
Step 145: after having reacted, is transferred to gel on sand core funnel, washes away unreacted sodium cyanoborohydride with redistilled water；
Step 146: wash gel with the acetate buffer solution that concentration is 0.2M that the sodium chloride of 2M, PH are 4.0 and the carbonic acid buffer that concentration is 0.1 that PH is 9.0 respectively, after being washed till neutrality with redistilled water, it is placed in three (methylol) aminomethane buffer solution that concentration is 0.2M that PH is 7.4, add preservative Sodium Azide, put in 4 DEG C of refrigerators and preserve.
The new method of a kind of protein immobilization the most according to claim 3, it is characterised in that: the also phosphoric acid buffer of the acetate buffer solution in described step 141.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610310137.6A CN105968207A (en) | 2016-05-12 | 2016-05-12 | Novel method for immobilizing protein |
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| CN201610310137.6A CN105968207A (en) | 2016-05-12 | 2016-05-12 | Novel method for immobilizing protein |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232988A (en) * | 2013-04-04 | 2013-08-07 | 山东大学（威海） | Agarase immobilization method |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232988A (en) * | 2013-04-04 | 2013-08-07 | 山东大学（威海） | Agarase immobilization method |
Non-Patent Citations (1)
| Title |
|---|
| 赵明等: "《介绍一种蛋白质固定化的新方法——氰基硼氢化钠还原法》", 《生命的化学（中国生物化学会通讯）》 * |
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