CN1621122A - Preparation of commodity type affinity column and its operation mode - Google Patents
Preparation of commodity type affinity column and its operation mode Download PDFInfo
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- CN1621122A CN1621122A CN 200310115050 CN200310115050A CN1621122A CN 1621122 A CN1621122 A CN 1621122A CN 200310115050 CN200310115050 CN 200310115050 CN 200310115050 A CN200310115050 A CN 200310115050A CN 1621122 A CN1621122 A CN 1621122A
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- affinity column
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Abstract
The present invention relates to the application of antibody capable of distinguishing heat shock protein (HSP) in preparing commercial affinity resin and affinity chromatographic column, and aims at extracting HSP and heat shock protein-polypeptide complexes formed with the HSP and non-covalent combined polypeptide antigen molecules fast, effectively and specifically from protein mixing liquid.
Description
Technical field
The invention belongs to biological technical field
Technical background
Difference by the various physicochemical properties between the molecule reaches comes out to need a series of very complicated and loaded down with trivial details operating process of experience to single albumen purifying from tissue extract or albumen miscellaneous solution, expends time in to grow and ultimate yield is very low.Along with the development of biochemical technology, a kind of easy and simple to handle, method---affinity chromatography of separating the high separation and purification biomacromolecule of specificity appears.Its principle is to utilize specific recognition and bonded characteristic between the Ag-Ab to come " single stage method " purification of target albumen.Its embodiment be can discern with the proteic antibody coupling of combining target to solid-phase resin, resin commonly used has the sepharose of cyanogen bromide-activated, as Sepharose 4B etc.When the used coupling resin of antibody when contacting with foreign protein solution, have only the albumen of being discerned and combining by antibodies specific ground just can be trapped on the resin in the solution, remove not combined non-specific foreign protein through flushing, use elutriant to dissociate at last and just can will be separated with other foreign protein with the target protein of antibodies by the albumen of antibodies.Utilize this principle and method " single stage method " separation and purification from tissue extract or foreign protein solution to go out needed target protein.It is easy and simple to handle utilizing affinity chromatography to come the macromolecular advantage of purifying biological, specificity height, good separating effect, be a kind of be laborsaving timesaving again efficient separation method.The affinity chromatography technology has now become a kind of proteic common method of separation and purification that is used in the Biochemical Lab.
Summary of the invention
The commodity-type affinity column that the present invention relates to prepare is in order to solve " single stage method " extraction HUMAN HEAT SHOCK PROTEINS (Heatshock protein is called for short HSP) from tissue extract or foreign protein solution with filler with becoming capo.Heat shock protein(HSP) is the intracellular family protein that is present in that a class homology is high, sequence is very conservative, can be divided into a plurality of members according to its molecular size and structural performance, as HSP-27, HSP-40, HSP-60, HSP-70, HSP-90, HSP-110, mainly be present in the endochylema, gp96 albumen mainly is present in the endoplasmic etc.They are in low-level expression status at ordinary times, in case the expression of HSP just can be increased sharply when sudden change of physiological, pathologic or environment appears in cell, so heat shock protein(HSP) be otherwise known as " stress protein ".Growth, differentiation, genetic transcription and the self-protection of assembling, transmembrane transport and the antigen presentation pair cell of HSP by participating in combination of proteins, folding, subunit play a significant role.Another key property of HSP is that it has the ability in conjunction with variant protein matter and polypeptide in the cell.They with cell in variant protein, polypeptide combines, participates in antigen presentation and its HSP itself does not produce antigenicity, so HSP be otherwise known as " molecular chaperones ".Prepare and a kind ofly can make that biology laboratory and clinical medicine center are quick, convenient to be gone out the proteic affinity column of HSP with separation and purification efficiently and become capo that crucial meaning is arranged in actual applications with filler or affinity chromatography.
Embodiment
1. prepare the affinity column filler:
Buy activatory sepharose resin, as CNBr-Sepharose 4B.
The coupling pre-treatment:
Taking by weighing 1g CNBr-Sepharose 4B fully embathes the expansion gel resin with 1mMo/L hydrochloric acid and uses coupling buffer again (0.1Mol/L NaHCO3,0.1Mol/L Na2CO3 and 0.5Mol/LNaCl pH8.5) wash 2 times.
Coupling:
The gel resin that disposes contains 10 μ Mol/LHSP-60 antibody with isopyknic coupling buffer and mixes, and at room temperature gentleness is shaken and mixed after 2 hours, discards solution and keeps gel resin.
Seal unnecessary binding site:
In gel resin, add 0.2Mol/L glycine solution 10ml, under the room temperature rotation mix 1 hour with on the sealing Sepharose 4B not with the site of antibodies.(0.1Mol/L NaCH2COOH and 0.5Mol/L NaCl pH4.0) alternately wash 2 times, use 100ml 0.15Mol/L PBS damping fluid (pH7.2) to clean gel resin lightly 2 times again with coupling buffer and acetate buffer.Affine resin can be kept supplying sample and do compatible reaction.
Rotproofing:
The affine resin such as the need that prepare are placed for some time, and resin should use the PBS that contains 1/10000 sodium azide to be immersed in and drop removes to put into after the excessive solution packing bag or bottle 4 ℃ of airtight preservations.
The packing of affinity chromatography column packing:
Will with per 5 grams of HSP-60 link coupled sepharose resin enclose seal in the aluminium plastic packaging bags or the brown vial of packing in approach the gland seal bottleneck with amino plug jam-pack and with aluminium.
Preserve:
The sepharose resin of the good antibody of coupling is sure not freezing, is kept in 4 ℃ the refrigerator stable performance within 1 year.
2. preparation affinity column:
The sepharose of the antibody coupling of 5g and anti-people HSP-60 is soaked with 100ml 0.15Mol/LPBS damping fluid (pH7.2) and install in the suitable chromatography column of size, prevent that the post bed from containing bubble.Use 0.15Mol/L PBS damping fluid (pH7.2) balance chromatography column.This post can be for the affine sample of going up.
The preservation of chromatography column:
The anticorrosion PBS damping fluid that use contains 1/10000 sodium azide is crossed post.Keep suitable preservative solution soaks resin and use diaphragm seal chromatography post in post two ends up and down to prevent resin stain and drying.The affinity column for preparing packed into separately to be kept in 4 ℃ of refrigerators after the sealing in aluminium plastic membrane or the Plastic film Bag, and validity period is 1 year.Packaged affinity column is sure not freezing.
Embodiment 1:
The preparation of affinity column:
Get 1 the bag 5g the Sepharose 4B affinity chromatography column packing of coupling HSP-60 antibody put into lid the bottle, with sucking-off excessive solution after 50ml 0.15Mol/L PBS (pH7.2) PBS damping fluid (the being called for short the PBS damping fluid) soaking and washing, resin can supply pending compatible reaction.
The preparation of tissue juice:
Take out tumor mass on one's body from rat, repair healthy tissues, the tumor tissues that cuts into fine grained chippings is put into-20 ℃ of refrigerator and cooled freeze and it is inserted homogenizer after 2 hours and grind.In mill, add the PBS damping fluid of 20ml precooling gradually and continue grinding.Lapping liquid is got supernatant liquor through refrigerated centrifuge 8000rpm 10 minutes after centrifugal and is done compatible reaction.
Compatible reaction:
Grinding supernatant liquor after centrifugal is transplanted in the bottle that the affinity chromatography resin extender is housed, tighten bottle cap and prevent a bottle interior solution leakage, affine resin mixed in bottle with the tumor tissues lapping liquid and be placed on and in 4 ℃ environment, leniently shake concussion on wobbler or the oscillator and spend the night, reach the antibody and the well-bound purpose of the antigen in the solution that make on the affine resin.
The dress post:
All be loaded into resin in the bottle and solution in the clean empty chromatography column next day, allows liquid flow flow out from the chromatography column bottom through resin, treats that liquid level goes out to add the PBS damping fluid when just having supported the resin face again and fully washes the ultraviolet absorption value OD of affinity column until effluent liquid
280＜0.02, reach the purpose that the nonspecific proteins flushing is removed.
The wash-out affinant:
With in glycine-hydrochloric acid soln (pH2.4) wash-out affinity column of 0.1Mol/L with the target protein of HSP-60 antibodies, i.e. HSP-60 albumen.The elution fraction of collecting uses 0.1Mol/L Tris-Cl damping fluid (pH8.0) neutralization in order to avoid protein denaturation rapidly.
The evaluation of isolate:
The purity of utilizing HSP-60 albumen usable highly effective liquid chromatography (HPLC) that the method for affinity chromatography is purified into or polyacrylamide gel electrophoresis (SDS-PAGE) to analyze eluted product, available ultraviolet spectrophotometry is done the mensuration of protein content.
The regeneration of post:
0.1Mol/L Tris-HCl with 10 column volumes contains 0.5Mol/L NaCl (pH8.5) damping fluid, and pH4.5,0.1mol/L sodium-acetate buffer cleans affinity column 2 times in turn, again with being kept at behind the abundant balance chromatography column of PBS damping fluid in 4 ℃ of refrigerators for using next time.
Affinity column can reuse after regenerating, and every affinity column iterative regenerable uses repeatedly.
Embodiment 2:
Take 1 the Sepherose 4B affinity column of coupling HSP-60 antibody wash balance to keep supplying sample with 0.15Mol/L PBS damping fluid (pH7.2) stream.
The pretreatment:
The foreign protein mixed solution that will contain the HSP-60 molecule is used PBS damping fluid dialysed overnight in 4 ℃ environment.
Compatible reaction:
The good foreign protein solution of will dialysing next day is added on the affinity column that balance got well, and keeps the flow velocity at 0.5ml/min.Treat to wash the ultraviolet absorption value OD of affinity column with a large amount of PBS streams after sample flow is finished until effluent liquid
280＜0.02.
The wash-out affinant:
Use the target protein of 0.1Mol/L glycine-HCl (pH2.4) wash-out and resin coupling antibodies, i.e. HSP-60 albumen.Collection contain eluate stream part and immediately in 4 ℃ environment to PBS damping fluid dialysed overnight.Wherein change the PBS damping fluid 1 to 2 time.
The purification Identification product:
Transfer to the solution in the dialysis tubing in the bottle next day.The product that utilizes the method separation and purification of affinity chromatography to come out can carry out protein concentration, purity and active mensuration as required.
The regeneration of affinity column:
0.1Mol/L Tris-HCl with 10 column volumes contains 0.5Mol/L NaCl (pH8.5) damping fluid, and pH4.5, and the 0.1mol/L sodium-acetate buffer cleans affinity column 2 times in turn, uses pH7.2 again, the abundant balance chromatography column of 0.15mol/L PBS.
Store:
The PBS damping fluid of the sodium azide of use adding 1/0,000 soaks resin and is stored in 4 ℃ of refrigerators.
Separated product is identified:
The HSP-60 albumen that the collection separation and purification goes out can adopt methods such as SDS-PAGE agarose gel electrophoresis, high performance liquid chromatography (HPLC), Western Blot, ultraviolet spectrometry to detect purity of protein and the content of separating.
Conclusion:
The HSP-60 albumen that contains low levels in foreign protein solution extracts the HSP-60 protein-specific ground concentration and separation in the foreign protein solution to be come out through " single stage method " of affinity column.This preparation coupling the method for affinity column of HSP antibody will can be used for preparing the proteic affinity column of commercial extraction HSP, reach effectively that separation and purification specifically goes out the proteic purpose of HSP from contain the proteic foreign protein solution of small amount of H SP.The HSP affinity column of this commodity-type can greatly make things convenient for and shorten the proteic program of user's separation and Extraction HSP of biology laboratory and medical institutions.
When using operation can according to but needn't stick to above experiment condition and method carry out the sample pretreatment of affinity chromatography, coupling, sealing, on sample, cleaning, wash-out etc.
Can will can discern and be coupled on the resin, mix as monoclonal antibody and be coupled on the Sepharose 4B resin the monoclonal antibody of HSP-70 and gp96 in conjunction with single HSP albumen or the proteic single or multiple mono-clonal of multiple HSP or polyclonal antibody.Use different HSP antibody couplings to affine resin, can be prepared into commodity-type affinity chromatography column packing or the affinity column that is fit to various needs as required.
Claims (8)
1. prepare a kind of can be quick from foreign protein solution, easy with extract the employed resin extender of the proteic affinity column of people HSP-60 specifically and become capo.
2. the resin extender for preparing this affinity column can be any can discern specifically and in conjunction with the proteic resin of HSP-60.
3. can be coupling can discern the resin that uses of this affinity chromatography column packing and in conjunction with proteic one or more antibody of HSP-60, they can be monoclonal antibody or polyclonal antibody.
The resin that uses of this affinity chromatography column packing not only coupling anti-people or mammal HSP-60 protein antibodies, the also identification of coupling simultaneously and in conjunction with other proteic antibody in people or the mammal heat-shock protein family, as proteic antibody such as HSP-27, HSP-40, HSP-70, HSP-90, HSP-110, gp-96.They can be mono-clonal or polyclonal antibody.
The resin that uses of affinity chromatography column packing can be coupling can discern with in conjunction with single a kind of or different several proteic antibody arbitrarily in people or the mammal heat-shock protein family, used antibody can be mono-clonal or polyclonal antibody.
6. the affine resin finished of preparation can directly use the container or the diaphragm seal packing of cracky not, anti-drying, lucifuge and be kept in the low temperature environment, they will offer the user and directly be filled in the post with the form of commodity, make things convenient for them to prepare affinity column.
But the affine resin finished of preparation can be filled in the bottom barrier resins but solution can effusive container in.This container can be any material and makes, and outer tube, transfer pipet, chromatography column that also can use syringe etc. made.
8. the affinity column that is prepared from should use protection against the tide, lucifuge, the film or the box of cracky do not pack with anti-drying, pollution.Packaged affinity column should be placed in 4 ℃ of cryogenic environment preservation and will directly offer the user with the form of commodity and use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310115050 CN1621122A (en) | 2003-11-24 | 2003-11-24 | Preparation of commodity type affinity column and its operation mode |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200310115050 CN1621122A (en) | 2003-11-24 | 2003-11-24 | Preparation of commodity type affinity column and its operation mode |
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| CN1621122A true CN1621122A (en) | 2005-06-01 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104880357A (en) * | 2014-02-28 | 2015-09-02 | 中国科学院过程工程研究所 | Preparation method of quartz capillary affinity chromatography column |
| CN107855115A (en) * | 2017-12-11 | 2018-03-30 | 杭州华安生物技术有限公司 | Affinity column, preparation method and application |
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
-
2003
- 2003-11-24 CN CN 200310115050 patent/CN1621122A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104880357A (en) * | 2014-02-28 | 2015-09-02 | 中国科学院过程工程研究所 | Preparation method of quartz capillary affinity chromatography column |
| CN107855115A (en) * | 2017-12-11 | 2018-03-30 | 杭州华安生物技术有限公司 | Affinity column, preparation method and application |
| CN107855115B (en) * | 2017-12-11 | 2020-06-02 | 杭州华安生物技术有限公司 | Affinity chromatographic column, preparation method and application |
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
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