CN110514821A - A kind of protein immunoblotting film is lived again liquid and its application method - Google Patents
A kind of protein immunoblotting film is lived again liquid and its application method Download PDFInfo
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- CN110514821A CN110514821A CN201910429201.6A CN201910429201A CN110514821A CN 110514821 A CN110514821 A CN 110514821A CN 201910429201 A CN201910429201 A CN 201910429201A CN 110514821 A CN110514821 A CN 110514821A
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- 239000007788 liquid Substances 0.000 title claims abstract description 68
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 51
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 51
- 238000003119 immunoblot Methods 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 239000012528 membrane Substances 0.000 claims description 16
- 229920002981 polyvinylidene fluoride Polymers 0.000 claims description 15
- 239000007790 solid phase Substances 0.000 claims description 8
- 238000010025 steaming Methods 0.000 claims description 8
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 4
- 239000002033 PVDF binder Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 2
- 229940127121 immunoconjugate Drugs 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims 8
- 238000003756 stirring Methods 0.000 claims 2
- 239000007864 aqueous solution Substances 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 claims 1
- 238000012546 transfer Methods 0.000 abstract description 3
- 238000001962 electrophoresis Methods 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 description 18
- 239000006166 lysate Substances 0.000 description 7
- 238000004140 cleaning Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 101710169105 Minor spike protein Proteins 0.000 description 1
- 101710081079 Minor spike protein H Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002796 luminescence method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
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- Physics & Mathematics (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Invention provides one kind can quickly and efficiently remove the primary antibody and secondary antibody combined on protein immunoblotting film, and protein immunoblotting film is lived again liquid and its application method.And in the case where the protein for not needing to re-start the transfer that electrophoresis, transferring film are not destroyed on film, protein immunoblot film may be reused.
Description
Technical field
The present invention relates to antibody test fields, specifically, being related to protein immunoblotting technology.Especially protein is exempted from
The recycling of epidemic disease blotting membrane.
Background technique
Protein immunoblotting method is the one kind to grow up in gel electrophoresis and solid-phase immunoassay technical foundation
Technology is widely used in the detection and identification of protein expression level.Its step includes: (1) gel electrophoresis of protein；(2) electric
The method of trace is by the Protein transfer in gel to solid phase carrier, on pvdf membrane；(3) spy is had on primary antibody and solid phase carrier
The antigen for determining antigenic determinant is specifically bound；(4) the labeled secondary antibody of HRP enzyme and an anti-binding；(5) second level is anti-
Body marker occur chemiluminescence reaction detection test antibodies molecular weight and expression quantity whether be expected to be consistent.
Under normal circumstances, the solid phase carrier for combining protein or antigen can only carry out a protein immunoblotting reality
It tests.On the basis of Immunology, the combination of antigen-antibody is that the non-covalent bond of molecular surface combines, and the compound of formation is not
Firmly, (such as low PH, high salt concentration) can be dissociated under certain condition.The present inventor is not influencing signal strength or weakness of developing
Under principle, this primary antibody and secondary antibody that can quickly and efficiently remove and combine on protein immunoblotting film has been invented, and
It does not need to re-start electrophoresis, transferring film, in the case where the protein for not destroying the transfer on film, makes protein immunoblot film can be with
The protein immunoblotting film of reuse is lived again liquid and its application method.
Summary of the invention
The present invention is that a kind of protein immunoblotting film of highly effective is lived again liquid.
Protein immunoblotting film provided by the present invention is lived again liquid, is made of A liquid and two component of B liquid.
The content for the component that the A liquid includes are as follows:
1% Coomassie brilliant blue,
35%-45% methanol solution,
10% acetic acid solution,
The double steaming solution of 44%-54%；
The content for the component that the B liquid includes are as follows:
95%-100% methanol solution,
0%-5% double steaming solution.
Protein immunoblotting film of the present invention liquid of living again can be carried out by following technical process, be included the following steps:
The preparation of the A liquid: (1) measuring 400 ml double steaming solutions, and the first of 100 ml acetic acid solutions and 350 ml-450 ml is added
Alcoholic solution；(2) 10g Coomassie brilliant blue powder is weighed, is added in the solution of step (1) and makes it dissolve, mix well；(3) with double
Steam the water liquid capacity-fixed that obtains step (2) to 1L, mix well protein immunoblotting film is lived again liquid A liquid, be stored at room temperature.
The preparation of the B liquid: (1) 0 ml-50 ml double steaming solution is measured；(2) with 100% methanol solution by step
(1) liquid capacity-fixed is to 1L, mix well protein immunoblotting film is lived again liquid B liquid, be stored at room temperature.
What the present invention was previously mentioned live again liquid A liquid provides a kind of suitable dissociation environment for antigen antibody complex, in A liquid
Excessive coomassie and all proteins in environment by ionic bond or hydrophobic effect in conjunction with, primary antibody secondary antibody is replaced, is reached
The purpose sufficiently dissociated to antigen antibody complex.Antigen or antibody molecule after dissociation, still keep original physicochemical property and
Biological character.
The effect of what the present invention was previously mentioned live again liquid B liquid is to elute A liquid from immunoblotting film.
Protein immunoblotting film provided by the invention liquid of living again mainly has following advantage.
1, the protein immunoblotting film for having transferred more histiocyte lysate samples can be made to be reused many times.
2, for the stability of experiment, the present invention can reduce experimental procedure, do not need loading, run glue and transferring film etc.
Experimental procedure, with same protein immunoblotting film with Various Tissues detecting to different antibodies, give the experiment side of bringing
Just.
3, it can be adapted for the Western Immuno divided by PVDF membrane (pvdf membrane) for protein solid phase carrier
The primary antibody and secondary antibody combined on blotting membrane.
4, the protein on solid phase carrier can be made to generate chromogenic reaction, sightless protein band originally can be dyed
Macroscopic blue is finally washed away with B liquid again.
5, for production cost, the present invention has greatly saved experiment consumptive material and human resources,
6, the primary antibody secondary antibody conjugate that liquid removal is incorporated on protein immunoblotting film of living again mentioned by the present invention needs 10
min-60 min.The reaction time of protein immunoblotting film and A liquid B liquid is best with 5 min-30 min.
Hereinafter, will in conjunction with specific embodiment, to technical solution of the present invention and advantage make more detailed explanation and
Explanation.It should be understood that the content presented in specification, specific embodiment and Figure of description, just to more
Technical solution of the present invention and its advantage are clearly demonstrated, protection scope of the present invention is not construed as limiting.Art technology
Personnel can on the basis of specification disclosure, for it is various it is reasonable changed after technical solution, as long as
Spirit of the invention is not departed from, the technical solution after various change is included within protection scope of the present invention.
Detailed description of the invention
Fig. 1 be transferred more Tissue lysates pvdf membrane combination Mouse anti-Tubulin-beta antibody first
Secondary developing result；
Fig. 2 be transferred more Tissue lysates pvdf membrane for the first time live again after combine Mouse anti-IDE antibody development knot
Fruit；
Fig. 3 be transferred more Tissue lysates pvdf membrane live again for the second time after combine Mouse anti-Cyclin H antibody
Developing result；
Fig. 4 be transferred more Tissue lysates pvdf membrane third time live again after combine Mouse anti-FLII antibody development
As a result
Specific embodiment
Coomassie brilliant blue used in preparation of reagents is purchased from Sigma-Aldrich.
Acetic acid solution, methanol solution used in preparation of reagents are purchased from Chinese Medicine group.
Solid phase carrier-pvdf membrane used in experimental procedure is purchased from U.S. Millipore company.
Primary antibody used in experimental procedure is respectively as follows:
(1) Mouse anti-Tubulin-beta, the monoclonal antibody of the anti-Tubulin-beta albumen of mouse, article No.: 66240-1-Ig knows
The molecular weight of other target protein: 50kDa comes from U.S. Proteintech company.
(2) Mouse anti-IDE, the monoclonal antibody of the anti-IDE albumen of mouse, article No.: 67106-1-Ig identifies point of target protein
Son amount: 100-120kDa comes from U.S. Proteintech company.
(3) the monoclonal antibody article No. of the anti-Cyclin H protein of Mouse anti-Cyclin H mouse: 67065-1-Ig identifies target
The molecular weight of albumen: 36kDa comes from U.S. Proteintech company.
(4) the monoclonal antibody article No. of the anti-FLII albumen of Mouse anti-FLII mouse: 67039-1-Ig identifies point of target protein
Son amount: 140-150kDa comes from U.S. Proteintech company.
Secondary antibody used in experimental procedure: the sheep anti-Mouse of HRP-Goat anti-Mouse horseradish peroxidase-labeled
Secondary antibody be purchased from U.S. Jackson ImmunoResearch company
ECL chromogenic substrate used in experimental procedure comes from U.S. Proteintech company.
Other experimental raws and reagent are laboratory conventional raw material and reagent.
1, experiment reagent is prepared
Protein immunoblotting film used in this experiment is lived again liquid, is made of A liquid and two component of B liquid.The group that the A liquid includes
The content divided are as follows:
1% Coomassie brilliant blue,
40% methanol solution,
10% acetic acid solution,
49% double steaming solution；
The content for the component that the B liquid includes are as follows:
100% methanol solution,
The liquid of living again of protein immunoblotting film used in this experiment can be carried out by following technical process, include the following steps: institute
It states the preparation of A liquid: (1) measuring 400 ml double steaming solutions, the methanol solution of 100 ml acetic acid solutions and 400ml is added；(2)
10g Coomassie brilliant blue powder is weighed, is added in the solution of step (1) and makes it dissolve, mix well；(3) with distilled water by step
(2) liquid capacity-fixed obtained to 1L, mix well protein immunoblotting film is lived again liquid A liquid, be stored at room temperature.
The preparation of the B liquid: the methanol for measuring 1L 100% is that protein immunoblotting film is lived again liquid B liquid, and room temperature is protected
It deposits.
The cleaning solution: volume ratio is the 10mM phosphate buffer of 0.05%Tween 20
The confining liquid: mass ratio is the cleaning solution of 5% skim milk.
2, experimental method
(1) Hela, HEK-293, HepG2, Jurkat, HSC-T6, MCF-7, NCCIT, HT-1080, LNCAP nine are used respectively
A cell line lysate carries out polyacrylamide gel electrophoresis, and albumen applied sample amount is 50 holes μ g/；
(2) method of the electroblotting of the albumen in gel is transferred on pvdf membrane, is closed with confining liquid；
(3) the diluted primary antibody (Mouse anti-Tubulin-beta) to be measured of 1:200000 confining liquid, incubation at room temperature 1.5 is added
Hour；
(4) cleaning solution washs primary antibody three times, and 10 minutes every time；
(5) the diluted ELIAS secondary antibody of 1:10000 confining liquid (HRP-Goat anti-Mouse) is added, is incubated at room temperature 1.5 hours；
(6) ELIAS secondary antibody five times (HRP-Goat anti-Mouse) of cleaning solution washing, 10 minutes every time；
(7) developed with ECL substrate luminescence method, developing result is shown in figure one, figure one: having transferred the pvdf membrane of more Tissue lysates
Develop for the first time, the Mouse anti-Tubulin-beta molecular weight measured as the result is shown is 50kDa, with theoretical molecular weight
Size be consistent.
(8) A liquid is mixed with used protein immunoblotting film in step (7), is placed on shaking table, is incubated at room temperature
It 5min-30min minutes, dries stand-by；
(9) B liquid is mixed with used protein immunoblotting film in step (8), is eluted to nothing on protein immunoblotting film
Blue bands；
(10) cleaning solution washs B liquid three times, and 1 minute every time；
(11) protein immunoblotting film is carried out room temperature with confining liquid to close 1 hour, cleaning solution washs 3 times；
(12) it is added and uses the diluted primary antibody of confining liquid 1:10000 (Mouse anti-IDE), be incubated at room temperature 1.5 hours；
(13) the same step of experimental method (4) (5) (6) (7), developing result are shown in that figure two, figure two illustrate: the PVDF after living again for the first time
Film developing result shows that the Mouse anti-IDE molecular weight measured is 110kDa, within the scope of theoretical molecular size range.Development
50kDa does not have band nearby as the result is shown, it was demonstrated that the primary antibody Mouse anti-Tubulin-beta being incubated for for the first time is washed completely
It takes off.
(14) the same step of experimental method (8) (9) (10) (11)；
(15) addition presses the diluted primary antibody of 1:10000 (Mouse anti-Cyclin H) with confining liquid, is incubated at room temperature 1.5 hours；
(16) the same step of experimental method (4) (5) (6) (7), developing result are shown in that figure three, figure three illustrate: pvdf membrane after living again for the second time
Developing result.The Mouse anti-Cyclin H molecular weight measured as the result is shown is 36kDa, the size phase with theoretical molecular weight
Symbol.Developing result shows 50kd and 110kDa nearby all without band, it was demonstrated that the primary antibody Mouse anti-being incubated for for the first time
Tubulin-beta and second of primary antibody Mouse anti-IDE being incubated for are eluted completely with antibody protein bound on film
Fall.
(17) the same step of experimental method (8) (9) (10) (11)；
(18) it is added and uses the diluted primary antibody of confining liquid 1:10000 (Mouse anti-FLII), be incubated at room temperature 1.5 hours；
(19) the same step of experimental method (4) (5) (6) (7), developing result are shown in that figure four, figure four illustrate: pvdf membrane after living again for the third time
Developing result.The Mouse anti-FLII molecular weight measured as the result is shown is 150kDa, is consistent with the size of theoretical molecular weight.
Developing result show 50kd, 110kd, 36kDa nearby without band, it was demonstrated that it is preceding three times with antibody protein bound on film quilt
It washes away completely.
Claims (6)
- The liquid 1. a kind of protein immunoblotting film is lived again, is made of A liquid and two component of B liquid, it is characterised in that:The A liquid includes the component of following content:1% Coomassie brilliant blue,35%-45% methanol solution,10% acetic acid solution,The double steaming solution of 44%-54%；The B liquid includes the component of following content:95%-100% methanol solution,0%-5% steams aqueous solution.
- 2. liquid A liquid according to claim 1 of living again, it is characterised in that: by Coomassie brilliant blue powder, methanol solution, acetic acid Solution and distilled water mix in proportion, and stirring is melted completely to powder, are stored at room temperature.
- 3. liquid B liquid according to claim 1 of living again, it is characterised in that: mix methanol solution and double steaming solution in proportion It closes, stirs evenly, be stored at room temperature.
- 4. according to claim 1,2, liquid of living again described in 3, it is characterised in that: its application is divided into two steps, and the first step is soaked with A liquid Dsred protein immunoblotting film 5min-30min dries after completing chromogenic reaction；Second step, with B liquid washed protein Diagnosis of Sghistosomiasis Mark film washes away primary antibody secondary antibody conjugate from protein immunoblotting film.
- 5. according to liquid as claimed in claim 4 of living again, it is characterised in that: it can go be divided by PVDF membrane (pvdf membrane) The primary antibody and secondary antibody combined on the protein immunoblotting film of protein solid phase carrier.
- 6. liquid according to claim 4 of living again, it is characterised in that: it removes the primary antibody combined on protein immunoblotting film The chemoluminescence method for being applicable in but being not limited in protein immunoblotting result detection method with secondary antibody.
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Cited By (3)
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| CN111965371A (en) * | 2020-08-24 | 2020-11-20 | 上海雅酶生物医药科技有限公司 | Formula of western blotting membrane antibody stripping solution and preparation and use methods thereof |
| CN115372627A (en) * | 2022-08-12 | 2022-11-22 | 温州医科大学 | Immunity blotting membrane eluent and its using method |
| CN116298238A (en) * | 2022-09-08 | 2023-06-23 | 温州医科大学 | Immunoblotting membrane regeneration liquid and use method thereof |
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| CN115372627A (en) * | 2022-08-12 | 2022-11-22 | 温州医科大学 | Immunity blotting membrane eluent and its using method |
| CN115372627B (en) * | 2022-08-12 | 2025-05-27 | 温州医科大学 | Immunoblotting membrane elution solution and use method thereof |
| CN116298238A (en) * | 2022-09-08 | 2023-06-23 | 温州医科大学 | Immunoblotting membrane regeneration liquid and use method thereof |
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