CN109851663A - Method for purifying His-tag marked foot-and-mouth disease epitope protein antigen by nickel-combined nano magnetic beads - Google Patents
Method for purifying His-tag marked foot-and-mouth disease epitope protein antigen by nickel-combined nano magnetic beads Download PDFInfo
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- CN109851663A CN109851663A CN201910194689.9A CN201910194689A CN109851663A CN 109851663 A CN109851663 A CN 109851663A CN 201910194689 A CN201910194689 A CN 201910194689A CN 109851663 A CN109851663 A CN 109851663A
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- 230000005291 magnetic effect Effects 0.000 title claims abstract description 118
- 239000011324 bead Substances 0.000 title claims abstract description 84
- 102000036639 antigens Human genes 0.000 title claims abstract description 61
- 108091007433 antigens Proteins 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 29
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 title abstract 4
- 241000710198 Foot-and-mouth disease virus Species 0.000 title abstract 4
- 239000000427 antigen Substances 0.000 claims abstract description 56
- 239000006228 supernatant Substances 0.000 claims abstract description 28
- 239000003480 eluent Substances 0.000 claims abstract description 17
- 238000005406 washing Methods 0.000 claims abstract description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 138
- 229910052759 nickel Inorganic materials 0.000 claims description 69
- 239000000243 solution Substances 0.000 claims description 52
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 20
- 238000004140 cleaning Methods 0.000 claims description 13
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 12
- 239000007995 HEPES buffer Substances 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 12
- 238000000746 purification Methods 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 238000005119 centrifugation Methods 0.000 claims description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 10
- 238000010521 absorption reaction Methods 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 230000027455 binding Effects 0.000 abstract description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 230000005298 paramagnetic effect Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- -1 1min is adsorbed Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 210000000003 hoof Anatomy 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The invention provides a method for purifying a foot-and-mouth disease epitope protein antigen marked by His-tag by nickel-combined nano magnetic beads, which comprises the following steps: putting 50 μ L of nickel-NTA nanometer magnetic beads into a centrifuge tube, adding 500 μ L of binding solution, mixing for 10 times, adsorbing for 1min by a magnetic frame, removing and discarding supernatant, and washing for 1 time; adding 1mL of binding solution and 100 mu L of His-tag marked foot-and-mouth disease epitope protein antigen into the centrifuge tube, mixing and reacting for 15min at 4 ℃, adsorbing for 1min by a magnetic frame, removing and discarding supernatant; adding 1mL of washing solution, mixing well, standing at room temperature for 5min, adsorbing for 2min by a magnetic rack, removing and discarding supernatant, and repeating for 1 time; adding 200 μ L of eluent, mixing at 4 deg.C for 10min, and adsorbing with magnetic frame for 2min to obtain purified and enriched His-tag labeled foot and mouth disease epitope protein antigen supernatant. The nickel-NTA nano antibody magnetic bead method for purifying the antigen has the advantages of high yield, high antigen concentration, easiness in downstream application and the like.
Description
Technical field
The present invention is the method for the aftosa neoepitope Western antigen that nickel closes nanometer magnetic beads for purifying His-tag label, is belonged to anti-
Former technical field of purification.
Background technique
Nickel-NTA nanometer magnetic bead because its with large specific surface area, adsorption capacity is strong, be easy to automate, operation quickly, extract
The more and more extensive purifies and separates for His-tag albumen of advantages such as efficient.When common splitter method is because of operation needed for it
Between the long, antigen concentration that obtains and yield it is low etc. insufficient and increase production cost, result in waste of resources.
Summary of the invention
In view of the deficienciess of the prior art, it is an object of the present invention to provide nickel to close nanometer magnetic beads for purifying His-tag label
The method of aftosa neoepitope Western antigen, to solve the problems mentioned in the above background technology, His-tag label of the invention
Aftosa neoepitope Western antigen yield is high, and the dosage of nickel conjunction-NTA nanometer magnetic bead, section can be adjusted according to the content of antigen
Cost-saving.
To achieve the goals above, the present invention is to realize by the following technical solutions: nickel closes nanometer magnetic beads for purifying
The method of the aftosa neoepitope Western antigen of His-tag label, includes the following steps:
Step 1: taking the nickel conjunction-NTA nanometer magnetic bead of 50 μ L to be put into 1.5mL centrifuge tube, and 500 μ l combination liquid are added to
It in nickel conjunction-NTA nanometer magnetic bead, mixes 10 times, above-mentioned centrifuge tube is placed on magnetic frame and carries out absorption nickel conjunction-NTA nanometer magnetic bead,
1min is adsorbed, supernatant is removed and discard, is washed repeatedly 1 time, bottom nickel conjunction-NTA nanometer magnetic bead is retained；The step is in order to excellent
Change the combining environmental of nickel conjunction-NTA nanometer magnetic bead.
Step 2: 1mL combination liquid and 100 μ L His-tag the aftosa neoepitope Western antigen marked are added to step 1
In obtain in the centrifuge tube of nickel conjunction-NTA nanometer magnetic bead, 4 DEG C of hybrid reaction 15min are put into centrifuge tube on magnetic frame and adsorb
1min removes and discards supernatant, retains the Ag-Ab magnetic bead conjugate A of centrifugation bottom of the tube, which, which is added, combines liquid energy
The combination of the aftosa neoepitope Western antigen and antibody of His-tag label is enough advantageously promoted, 4 DEG C of low temperature can be protected preferably
The aftosa neoepitope Western antigen of His-tag label is not destroyed by environmental condition, sufficiently captures His- by above-mentioned antibody magnetic bead
The aftosa neoepitope Western antigen of tag label；
Step 3: being added Ag-Ab magnetic bead conjugate A obtained in 1mL cleaning solution washing step two, is uniformly mixed,
It is placed at room temperature for 5min, centrifuge tube is put on magnetic frame and adsorbs 2min, removes and discards supernatant, be repeated 1 times, obtain centrifuge tube
The Ag-Ab magnetic bead conjugate B of bottom；Cleaning solution, which is added, in the step can fully remove Ag-Ab magnetic bead conjugate A
The impurity of the non-specific binding on surface.
Step 4: 200 μ L eluents are added in the centrifuge tube for obtaining Ag-Ab magnetic bead conjugate B in step 3,
10min is mixed at 4 DEG C, centrifuge tube is put on magnetic frame and adsorbs 2min, the mouth hoof of the His-tag label after obtaining purification enrichment
Epidemic disease neoepitope Western antigen supernatant；The mouth that the purpose of eluent is the His-tag label of dissociation antibody magnetic bead capture is added in the step
Fever aphthous neoepitope Western antigen.
Further, the combination liquid is to contain 1-7mmol/L imidazole solution, 0.2-0.8mol/L NaCl solution, 10-
The buffer of 50mmol/L Tris-HCl pH=7-8 or the HEPES buffer solution of pH=7-8.
Further, the cleaning solution is to contain 20-30mmol/L imidazole solution, 0.1-0.4mol/L NaCl solution, 5-
The buffer of 30mmol/L Tris-HCl pH=7-8 or the HEPES buffer solution of pH=7-8.
Further, the eluent is to contain 200-700mmol/L solution, 0.1-0.4mol/L NaCl solution, 5-
The buffer of 30mmol/L Tris-HCl pH=7-8 or the HEPES buffer solution of pH=7-8.
Beneficial effects of the present invention:
(1) the nickel conjunction-NTA nanometer magnetic bead specific surface area of the invention orders of magnitude more several greatly than micron magnetic bead, unit volume
Adsorption capacity is more excellent；Nickel conjunction-NTA nanometer magnetic bead magnetic intensity is more several times greater than micron magnetic bead, therefore sample not easy to lose, is more suitable for automatic
Change operation；Operation of the present invention is simple, the operating time is short, yield is high, and having in nickel conjunction-NTA nanometer magnetic bead can be with His-tag
Nickel-NTA the element of the aftosa neoepitope Western antigen binding of label, the mouth hoof of nickel conjunction-NTA nanometer magnetic bead and His-tag label
Epidemic disease neoepitope Western antigen passes through affinity Non-covalent binding.
(2) the aftosa neoepitope Western antigen of nickel conjunction-NTA nanometer magnetic bead of the invention and His-tag label can be in liquid
It communicated mixing to come into full contact with, so that 2-5 times of capability improving of nickel conjunction-NTA nanometer magnetic bead capture antigen；Since nickel conjunction-NTA receives
Rice paramagnetic particle method does not need Filter column, therefore avoids the pretreatment of Filter column, the column volume and sample ratio of super large and substantially
The problem of long-pending eluent dilute sample；High salt concentration imidazoles is usually contained in eluent, lower step application before need dialyse or
Column chromatography removal imidazoles.The antigen concentration that nickel conjunction-NTA nanometer magnetic bead obtains is high, and the content of imidazoles can be reduced by dilution, and
Dialysis or column chromatography are not needed.
(3) present invention takes around 1 hour, and yield is than conventional high 3 times of splitter method or so；And nickel conjunction-NTA nano magnetic
Pearl does not need time-consuming pretreatment before use；Follow-on test and tracking eluent are not needed in antigen separation process；It can root
According to the dosage of the content adjustment nickel conjunction-NTA nanometer magnetic bead of antigen, cost is saved.
(4) nickel-NTA nano antibody paramagnetic particle method purifying antigen of the invention has yield high, and antigen concentration is big, is easy to downstream
Using etc. advantages.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is the electricity of the method for the aftosa neoepitope Western antigen that nickel of the present invention closes nanometer magnetic beads for purifying His-tag label
Swimming experiment schematic diagram.
Specific embodiment
To be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, below with reference to
Specific embodiment, the present invention is further explained.
Embodiment 1
It takes the nickel conjunction-NTA nanometer magnetic bead of 50 μ L to be put into 1.5mL centrifuge tube, by 500 μ l combination liquid, is added to nickel conjunction-
It in NTA nanometer magnetic bead, mixes 10 times, above-mentioned centrifuge tube is placed on magnetic frame and carries out absorption nickel conjunction-NTA nanometer magnetic bead, absorption
1min removes and discards supernatant, washes repeatedly 1 time, retains bottom nickel conjunction-NTA nanometer magnetic bead；By 1mL combination liquid and 100 μ L
The aftosa neoepitope Western antigen of His-tag label is added in the centrifuge tube of nickel conjunction-NTA nanometer magnetic bead, 4 DEG C of hybrid reactions
15min is put into centrifuge tube on magnetic frame and adsorbs 1min, removes and discard supernatant, retains the Ag-Ab of centrifugation bottom of the tube
Magnetic bead conjugate A is added 1mL cleaning solution and washs Ag-Ab magnetic bead conjugate A, is uniformly mixed, is placed at room temperature for 5min, will be from
Heart pipe, which is put on magnetic frame, adsorbs 2min, removes and discards supernatant, be repeated 1 times, and obtains the Ag-Ab magnetic of centrifugation bottom of the tube
200 μ L eluents are added in above-mentioned centrifuge tube, mix 10min at 4 DEG C, centrifuge tube is put on magnetic frame by pearl conjugate B
2min is adsorbed, the aftosa neoepitope Western antigen supernatant of the His-tag label after obtaining purification enrichment.
It is to contain 7mmol/L imidazole solution, 0.2mol/L NaCl solution, 10mmol/L in conjunction with liquid in the present embodiment
The buffer of Tris-HCl pH=7.
In the present embodiment, the HEPES buffer solution of cleaning solution pH=7.
In the present embodiment, eluent is to contain 700mmol/L solution, 0.4mol/L NaCl solution, 5mmol/L Tris-
The buffer of HCl pH=8.
Embodiment 2
It takes the nickel conjunction-NTA nanometer magnetic bead of 50 μ L to be put into 1.5mL centrifuge tube, by 500 μ l combination liquid, is added to nickel conjunction-
It in NTA nanometer magnetic bead, mixes 10 times, above-mentioned centrifuge tube is placed on magnetic frame and carries out absorption nickel conjunction-NTA nanometer magnetic bead, absorption
1min removes and discards supernatant, washes repeatedly 1 time, retains bottom nickel conjunction-NTA nanometer magnetic bead；By 1mL combination liquid and 100 μ L
The aftosa neoepitope Western antigen of His-tag label is added in the centrifuge tube of nickel conjunction-NTA nanometer magnetic bead, 4 DEG C of hybrid reactions
15min is put into centrifuge tube on magnetic frame and adsorbs 1min, removes and discard supernatant, retains the Ag-Ab of centrifugation bottom of the tube
Magnetic bead conjugate A is added 1mL cleaning solution and washs Ag-Ab magnetic bead conjugate A, is uniformly mixed, is placed at room temperature for 5min, will be from
Heart pipe, which is put on magnetic frame, adsorbs 2min, removes and discards supernatant, be repeated 1 times, and obtains the Ag-Ab magnetic of centrifugation bottom of the tube
200 μ L eluents are added in above-mentioned centrifuge tube, mix 10min at 4 DEG C, centrifuge tube is put on magnetic frame by pearl conjugate B
2min is adsorbed, the aftosa neoepitope Western antigen supernatant of the His-tag label after obtaining purification enrichment.
In the present embodiment, the HEPES buffer solution for being pH=8 in conjunction with liquid.
In the present embodiment, cleaning solution is to contain 20mmol/L imidazole solution, 0.1mol/L NaCl solution, 30mmol/L
The buffer of Tris-HCl pH=7.
In the present embodiment, eluent is the HEPES buffer solution of pH=8.
Embodiment 3
It takes the nickel conjunction-NTA nanometer magnetic bead of 50 μ L to be put into 1.5mL centrifuge tube, by 500 μ l combination liquid, is added to nickel conjunction-
It in NTA nanometer magnetic bead, mixes 10 times, above-mentioned centrifuge tube is placed on magnetic frame and carries out absorption nickel conjunction-NTA nanometer magnetic bead, absorption
1min removes and discards supernatant, washes repeatedly 1 time, retains bottom nickel conjunction-NTA nanometer magnetic bead；By 1mL combination liquid and 100 μ L
The aftosa neoepitope Western antigen of His-tag label is added in the centrifuge tube of nickel conjunction-NTA nanometer magnetic bead, 4 DEG C of hybrid reactions
15min is put into centrifuge tube on magnetic frame and adsorbs 1min, removes and discard supernatant, retains the Ag-Ab of centrifugation bottom of the tube
Magnetic bead conjugate A is added 1mL cleaning solution and washs Ag-Ab magnetic bead conjugate A, is uniformly mixed, is placed at room temperature for 5min, will be from
Heart pipe, which is put on magnetic frame, adsorbs 2min, removes and discards supernatant, be repeated 1 times, and obtains the Ag-Ab magnetic of centrifugation bottom of the tube
200 μ L eluents are added in above-mentioned centrifuge tube, mix 10min at 4 DEG C, centrifuge tube is put on magnetic frame by pearl conjugate B
2min is adsorbed, the aftosa neoepitope Western antigen supernatant of the His-tag label after obtaining purification enrichment.
In the present embodiment, the HEPES buffer solution for being pH=8 in conjunction with liquid.
In the present embodiment, cleaning solution is the HEPES buffer solution of pH=7.
In the present embodiment, eluent is the HEPES buffer solution of pH=8.
Embodiment 4
It takes the nickel conjunction-NTA nanometer magnetic bead of 50 μ L to be put into 1.5mL centrifuge tube, by 500 μ l combination liquid, is added to nickel conjunction-
It in NTA nanometer magnetic bead, mixes 10 times, above-mentioned centrifuge tube is placed on magnetic frame and carries out absorption nickel conjunction-NTA nanometer magnetic bead, absorption
1min removes and discards supernatant, washes repeatedly 1 time, retains bottom nickel conjunction-NTA nanometer magnetic bead；By 1mL combination liquid and 100 μ L
The aftosa neoepitope Western antigen of His-tag label is added in the centrifuge tube of nickel conjunction-NTA nanometer magnetic bead, 4 DEG C of hybrid reactions
15min is put into centrifuge tube on magnetic frame and adsorbs 1min, removes and discard supernatant, retains the Ag-Ab of centrifugation bottom of the tube
Magnetic bead conjugate A is added 1mL cleaning solution and washs Ag-Ab magnetic bead conjugate A, is uniformly mixed, is placed at room temperature for 5min, will be from
Heart pipe, which is put on magnetic frame, adsorbs 2min, removes and discards supernatant, be repeated 1 times, and obtains the Ag-Ab magnetic of centrifugation bottom of the tube
200 μ L eluents are added in above-mentioned centrifuge tube, mix 10min at 4 DEG C, centrifuge tube is put on magnetic frame by pearl conjugate B
2min is adsorbed, the aftosa neoepitope Western antigen supernatant of the His-tag label after obtaining purification enrichment.
It is to contain 1mmol/L imidazole solution, 0.8mol/L NaCl solution, 50mmol/L in conjunction with liquid in the present embodiment
The buffer of Tris-HCl pH=8.
In the present embodiment, cleaning solution is to contain 30mmol/L imidazole solution, 0.4mol/L NaCl solution, 5mmol/L
The buffer of Tris-HCl pH=8.
In the present embodiment, eluent is to contain 200mmol/L solution, 0.1mol/L NaCl solution, 30mmol/L Tris-
The buffer of HCl pH=7.
As shown in Figure 1, A and B is the result of secondary experiment after purification after purification in figure.From the graph it can be seen that the weight of test
Renaturation is fine, and only an apparent master tape, other miscellaneous bands are weaker after purification；It can be with according to the brightness of each band after purification
It extrapolates nickel and closes the purity that nanometer magnetic bead method can obtain the aftosa neoepitope Western antigen of about 90%His-tag label；Pass through
The brightness that comparison purifies forward and backward master tape can extrapolate the aftosa neoepitope Western antigen protein yield that His-tag is marked
40%.It in addition may be that other intracellular albumen containing histidine are non-specifically incorporated in nickel conjunction-than darker miscellaneous band in figure
On NTA nanometer magnetic bead, here it can be seen that the deficiency of nickel conjunction-NTA method itself.From experiment process, whole process of purification
It is simple and efficient.
Embodiment 5
The aftosa neoepitope Western antigen and nickel zygostyle that nickel is closed to the His-tag label of nanometer magnetic bead method purifying respectively purify
The aftosa neoepitope Western antigen of His-tag label compares, and see Table 1 for details.
1 nickel of table conjunction nanometer magnetic bead method purifying antigen is compared with nickel zygostyle purifying antigen
| Nickel closes paramagnetic particle method | Nickel column chromatography | |
| Rate of recovery of antigen | 35-45% | 5-15% |
| Obtained antigen concentration | 10-20% | 5-8% |
| Required antigen minimum volume | 10-30μL | 500-1000μL |
It as shown in Table 1, is returning for nickel zygostyle purifying antigen using the rate of recovery of antigen that nickel closes nanometer magnetic bead method purifying antigen
3-7 times of yield, and it is 2 times of the antigen concentration that nickel zygostyle obtains or more that nickel, which closes the antigen concentration that nanometer magnetic bead method obtains, is adopted
The 2-3% that antigen minimum volume needed for nanometer magnetic bead method is purified is nickel zygostyle is closed with nickel.
In conclusion nickel closes the antigen yield of nanometer magnetic bead method purifying 40% or so, than conventional nickel column chromatography
3-7 times high, the high 2 times or more of concentration, nickel, which closes nanometer magnetic bead method purifying antigen, has yield high, and antigen concentration is big, is easy to downstream
Using etc. advantages.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention, for this field skill
For art personnel, it is clear that invention is not limited to the details of the above exemplary embodiments, and without departing substantially from spirit of the invention or
In the case where essential characteristic, the present invention can be realized in other specific forms.Therefore, in all respects, should all incite somebody to action
Embodiment regards exemplary as, and is non-limiting, the scope of the present invention by appended claims rather than on state
Bright restriction, it is intended that including all changes that fall within the meaning and scope of the equivalent elements of the claims in the present invention
It is interior.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (4)
1. the method that nickel closes the aftosa neoepitope Western antigen of nanometer magnetic beads for purifying His-tag label, which is characterized in that including such as
Lower step:
Step 1: taking the nickel conjunction-NTA nanometer magnetic bead of 50 μ L to be put into 1.5mL centrifuge tube, by 500 μ l combination liquid, is added to nickel
It in conjunction-NTA nanometer magnetic bead, mixes 10 times, above-mentioned centrifuge tube is placed on magnetic frame and carries out absorption nickel conjunction-NTA nanometer magnetic bead, is inhaled
Attached 1min removes and discards supernatant, washes repeatedly 1 time, retains bottom nickel conjunction-NTA nanometer magnetic bead；
Step 2: 1mL combination liquid and 100 μ L His-tag the aftosa neoepitope Western antigen marked are added in step 1 and obtained
Into the centrifuge tube of nickel conjunction-NTA nanometer magnetic bead, 4 DEG C of hybrid reaction 15min are put into centrifuge tube on magnetic frame and adsorb 1min, move
Out and supernatant is discarded, retains the Ag-Ab magnetic bead conjugate A of centrifugation bottom of the tube；
Step 3: being added Ag-Ab magnetic bead conjugate A obtained in 1mL cleaning solution washing step two, is uniformly mixed, room temperature
5min is placed, centrifuge tube is put on magnetic frame and adsorbs 2min, removes and discards supernatant, be repeated 1 times, obtains centrifugation bottom of the tube
Ag-Ab magnetic bead conjugate B；
Step 4: 200 μ L eluents are added in the centrifuge tube for obtaining Ag-Ab magnetic bead conjugate B in step 3,4 DEG C
Lower mixing 10min, centrifuge tube is put on magnetic frame and adsorbs 2min, the aftosa of the His-tag label after obtaining purification enrichment
Neoepitope Western antigen supernatant.
2. the side that nickel according to claim 1 closes the aftosa neoepitope Western antigen of nanometer magnetic beads for purifying His-tag label
Method, which is characterized in that the combination liquid is to contain 1-7mmol/L imidazole solution, 0.2-0.8mol/L NaCl solution, 10-
The buffer of 50mmol/L Tris-HCl pH=7-8 or the HEPES buffer solution of pH=7-8.
3. the side that nickel according to claim 1 closes the aftosa neoepitope Western antigen of nanometer magnetic beads for purifying His-tag label
Method, which is characterized in that the cleaning solution is to contain 20-30mmol/L imidazole solution, 0.1-0.4mol/L NaCl solution, 5-
The buffer of 30mmol/L Tris-HCl pH=7-8 or the HEPES buffer solution of pH=7-8.
4. the side that nickel according to claim 1 closes the aftosa neoepitope Western antigen of nanometer magnetic beads for purifying His-tag label
Method, which is characterized in that the eluent is to contain 200-700mmol/L solution, 0.1-0.4mol/L NaCl solution, 5-
The buffer of 30mmol/L Tris-HCl pH=7-8 or the HEPES buffer solution of pH=7-8.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110346561A (en) * | 2019-06-26 | 2019-10-18 | 南京工业大学 | Preparation method and application of silicon dioxide magnetic microspheres for rapidly detecting anaphylactic reaction |
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