CN107271656B - A kind of protein immunoblotting membrane regeneration liquor - Google Patents
A kind of protein immunoblotting membrane regeneration liquor Download PDFInfo
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Abstract
The invention belongs to biology fields, are mainly concerned with a kind of regenerated liquid of protein immunoblotting film.This protein immunoblotting membrane regeneration liquor can efficiently remove primary antibody and secondary antibody on Western blotting film, the repetition for the same blotting membrane that displaced albumen in being detected for use in protein immunoblot efficiently utilizes, used blotting membrane can easily be re-used and detect other destination proteins, it is time saving and energy saving, and albumen loading and the error brought again can be eliminated, enhance comparativity.By the test experience of multiple antibody, this protein immunoblotting membrane regeneration liquor can usually reuse Western blotting film at least three times, molecular biology routine experiment protein immunoblotting is greatly saved and detects consumed correlative charges, and using this reagent the repetition that protein immunoblot film can be realized in about 30 minutes is only needed to reuse.It is and compatible with the direct Detection Method of substrate development process, chemiluminescence colour developing, fluorescent marker secondary antibody.
Description
Technical field
The present invention relates to a kind of protein immunoblotting membrane regeneration liquors, this protein immunoblotting membrane regeneration liquor can be quick
The primary antibody and secondary antibody combined on protein immunoblotting film is efficiently fully removed, makes protein immunoblot film can be with repetitive cycling
It uses.
Background technology
Proteomics (proteomics) refers to using all proteins of genome encoding as research object, from cell or
The composition and its changing rule of research protein in integral level are organized, so as to deeply recognize the various physiology and disease of organism
Reason.Compared with being studied with traditional protein, proteomics research embodies comprehensive, globality, high throughput, large-scale spy
Point.The research of proteomics carries out proof analysis with nothing for having completed the protein group of the theoretical prediction of genome plan
The important function that method substitutes.Proteomic techniques are complex, including three aspect of Separation of Proteins, identification and information analysis
Content.Wherein, the separation identification of target protein is one of core technology of proteomics.Western blotting, also known as protein
Trace (Western blot) is the side of certain protein in the specific binding detection of complex sample according to antigen-antibody
Method.Western blotting method is the protein using gel electrophoresis separating natural or denaturation first, then by the protein in gel
Electricity consumption immunoblot method is transferred on chemically inert macromolecule transfer membrane, afterwards by the specific primary antibodies (one of destination protein
It is anti-) identify with the destination protein on transfer membrane and be immunized association reaction, then with labeled secondary antibody (secondary antibody) and one
Anti-binding proves the presence of destination protein and the difference of expression quantity with to the detection of label in secondary antibody molecule afterwards.Antibody with
It is the mutual property of structure based on antigenic determinant (epitope) with the groove molecular surface of antibody hypervariable region that antigen, which can be specifically bound,
It is combined with affinity.It is antigen and the invertibity antigen primary antibody bluk recombination of specific bond formation under certain condition of corresponding antibody
The process of object.In western blot detection process, in addition to testing goal albumen, it is also necessary to detect TUBULIN, ACTIN etc.
The metastable protein of expression quantity is as with reference to the uniformity of the definite total applied sample amount of protein.It is examined in common western blot
During survey, carry the blotting membrane of albumen can only once do primary immune response trace detection, can not on same film simultaneously again
Internal reference albumen is detected, if to carry out the detection of internal reference albumen, protein electrophoresis transferring film etc. same one can only be carried out again
Serial protein immunization detection operation, not only time-consuming bothersome, albumen shape on blotting membrane caused by also resulting in repetition loading and operation
The change of state.So if not only can be with the expression quantity changing condition of testing goal albumen on same film, but also can detect
The metastable situation with reference to albumen or the other other albumen of detection of the expression quantity such as TUBULIN, ACTIN is optimal albumen
Immune-blotting method.
A kind of efficient protein matter Western blotting membrane regeneration liquor of the present invention can be detected in first time protein immunoblot and operated
Substantially efficiently removal is incorporated in primary antibody and secondary antibody on destination protein after being fully completed, and is detected with this for protein immunoblot
In displaced albumen same blotting membrane recycling, can simply and easily reuse very much blotting membrane detection it is other
Target protein is compared with the protein immunoblots operation sequence such as SDS-PAGE electrophoresis is re-started, not only time saving and energy saving, and can
To eliminate the protein systematic error that loading and electrophoresis band come again, internal reference albumen and destination protein are carried out on same film
Detection, so as to which comparativity is stronger.The utility model passes through the test experience of multiple antibody, can usually reuse blotting membrane 2
~3 times.And the reuse that protein immunoblot film can be realized in about 30 minutes is only needed using this kit.
Bibliography
The experimental study experimental technique of the protein immunoblottings technology such as Zhang Yanwan and 2008 (10) of management
The Western blots such as Zhao Junfang detection allergen specificity IgG great variety of modern medical examining magazine 2008 (01)
The content of the invention
One of the object of the invention is to provide a kind of can be simple and efficient and fully removes what is combined on protein immunoblotting film
The method of primary antibody and secondary antibody；
The two of the object of the invention are to provide a kind of can be simple and efficient and fully remove what is combined on protein immunoblotting film
The regenerated liquid of primary antibody and secondary antibody；
The three of the object of the invention, which are to provide, a kind of can be suitable for the primary antibody combined on NC films and pvdf membrane and secondary antibody again
Raw liquid；
The four of the object of the invention be to provide it is a kind of can be suitable for the bottom protein immunoblotting film signal detecting method
The direct Detection Method of object development process, chemoluminescence method and fluorescent marker secondary antibody can be simple and efficient fully removal Western Immuno
The primary antibody and the regenerated liquid of secondary antibody combined on blotting membrane；
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
The detection of primary immune response trace is can be only done for a film in the immune-blotting method (Western blot) of albumen
The problem of, present inventor has performed further investigations.On antibody and the theoretical foundation of antigen binding, i.e., antibody is all immune globulin
White and most antigens are also protein, and minority is the substances such as polysaccharide, lipoid, nucleic acid.Antigen has the spy of height with antibody response
The opposite sex.This reaction is the combination of molecular surface, although quite stable, because one antibody of antigen is not affected by destruction in itself, they
It is still separable.The combination of antigen-antibody is that the non-covalent bond of molecular surface combines, and the compound of formation is unstable, certain
Under the conditions of can dissociate, therefore antigen-antibody reaction formed compound process be a dynamic equilibrium.Antigen antibody complex
Dissociation depends on both sides factor：First, antibody corresponds to the affinity of antigen；Second is that influence of the environmental factor to compound.Cause
This can be quiet to the affinity of antigen or change environmental factor ionic strength and pH value destruction interionic by destroying antibody
Electric attraction reduces the combination power of antigen-antibody, promotes its dissociation.Therefore, in view of antibody and the invertibity mistake of antigen binding effect
Journey, the present invention are reacted by increasing the ionic strength in antibody and antigen binding environment using increase ionic strength and reduction
The hydrochloric acid guanidine salt and SDS salt and Tween 20 of pH in liquid promotes the separation of primary antibody and antigen, then cleans these and be unfavorable for
After the environmental factor that antibody antigen combines, so as to carry out the protein immunoblotting test experience of the second wheel.
Description of the drawings
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But these embodiments are only exemplary, are not limited the present invention in any way.Those skilled in the art should
Understand, can the details and form of technical solution of the present invention be modified or replaced under without departing from the scope of the present invention
It changes, but these modifications and in replacement all belongs to the scope of protection of the present invention.
1. experiment material：
1.1 murine liver tissue；
1.2 antibody
The polyclonal specific antibody A in rabbit source is purchased from Abcam companies of the U.S.；
The polyclonal specific antibody B in rabbit source is purchased from Abcam companies of the U.S.；
Mouse resource monoclonal specific antibody C is purchased from Abcam companies of the U.S.；
The polyclonal specific antibody TUBULIN in rabbit source is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；
Mouse resource monoclonal specific antibody β-ACTIN are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；
Sheep source non-specificity horseradish peroxidase-labeled HRP-Goat-anti-Rabbit secondary antibodies are purchased from China fir gold in Beijing
Bridge Bioisystech Co., Ltd；
Sheep source non-specificity horseradish peroxidase-labeled HRP-Goat-anti-Mouse secondary antibodies are purchased from Beijing Zhong Shan Golden Bridge
Bioisystech Co., Ltd；
Sheep source non-specific alkaline phosphatase mark AP-Goat-anti-Rabbit secondary antibodies are purchased from Beijing Zhong Shan Golden Bridge biology
Technology Co., Ltd.；
Sheep source non-specific alkaline phosphatase mark AP-Goat-anti-Mouse secondary antibodies are purchased from Beijing Zhong Shan Golden Bridge biology
Technology Co., Ltd.；
2. experiment reagent：
Tris salt：Beijing is glad through biotechnology Co., Ltd of section
DTT (dithiothreitol (DTT))：Beijing is glad through biotechnology Co., Ltd of section
SDS (lauryl sodium sulfate)：MP Biomedicals (Shanghai) Co., Ltd.
EDTA-Na2(disodium ethylene diamine tetraacetate)：MP Biomedicals (Shangha) Co., Ltd.
Acrylamide/methene acrylamide：Chinese medicines group chemical reagent Beijing Co., Ltd
Ammonium persulfate：Beijing is glad through biotechnology Co., Ltd of section
TEMED (N, N, N ', N '-tetramethylethylenediamine)：MP Biomedicals companies of the U.S.
Glycine：Chinese medicines group chemical reagent Beijing Co., Ltd
Pre-dyed standard molecular weight albumen：Biorad companies of the U.S.
Methanol：Chinese medicines group chemical reagent Beijing Co., Ltd
Glacial acetic acid：Chinese medicines group chemical reagent Beijing Co., Ltd
Sodium chloride：Chinese medicines group chemical reagent Beijing Co., Ltd
Potassium dihydrogen phosphate：Chinese medicines group chemical reagent Beijing Co., Ltd
Disodium hydrogen phosphate：Chinese medicines group chemical reagent Beijing Co., Ltd
Glycerine：Chinese medicines group chemical reagent Beijing Co., Ltd
Bromophenol blue：MP Biomedicals companies of the U.S.
Tween-20：MP Biomedicals companies of the U.S.
Bradford determination of protein concentration reagents：Biorad companies of the U.S.
NBT/BCIP substrate colour reagents：Sangon Biotech (Shanghai) Co., Ltd.
ECL- chemical luminous substrate kits：Bio Rad Laboratories
3. test consumptive material and instrument：
0.2 μm of nitrocellulose filter NC membrane apertures：PALL companies of the U.S.
0.2 μm of PALL FluoroTrans PVDF transfer films：PALL companies of the U.S.
Low-temperature and high-speed centrifuge：Sigma Co., USA
Albumen glue and electrophoresis system：Biorad companies of the U.S.
Wet type membrane-transferring device：Biorad companies of the U.S.
Las500 Image-forming instruments：GE companies of the U.S.
Azure C500 Image-forming instruments：Azure companies of the U.S.
4. the preparation of main agents：
4.1SDS-PAGE electrophoresis
10%APS：0.1g ammonium persulfates are weighed, deionized water dissolving is simultaneously settled to 1mL, and 4 DEG C preserve use in 1 week.
10%SDS：10gSDS is weighed, adds and is settled to 100mL from water.
1.5MTris-HCl(pH8.8)：It weighs 18.17g Tris salt, after deionized water dissolving, pH value is adjusted with dense HCl
To 8.8, finally 100mL, 4 DEG C of preservations are settled to deionized water.
0.5MTris-HCl(pH6.8)：Weigh 6.05g Tris salt, after deionized water dissolving, with dense HCl adjust pH to
6.8, finally 100mL, 4 DEG C of preservations are settled to deionized water.
5 × albumen sample-loading buffer：0.2g SDS, 0.07571g Tris, 5mL glycerine, 0.05g bromophenol blues, 0.01DTT,
5mL distilled water is distributed into 1mL aliquots, -20 DEG C of preservations after mixing with HCl tune pH6.8, constant volume to 10mL.
Electrophoretic buffer：15g Tris salt, 72g glycine, 5g SDS are weighed, deionized water is settled to 1000mL.
Transferring film buffer solution：3.028g Tris salt is weighed, after 14.414g glycine addition deionized water is settled to 800mL,
200mL methanol constant volumes are added to 1000mL.
1×TBS：Weigh 30.2g Tria salt, 8.766g sodium chloride, after addition deionized water is settled to 1000mL.
1×TBST：30.2g Tris salt is weighed, 8.766g sodium chloride after addition deionized water is settled to 1000mL, then adds
Enter 500 μ L Tween-20 mixings.
4.2 protein immunoblots detect
Transferring film buffer solution：3.028g Tris salt is weighed, after 14.414g glycine addition deionized water is settled to 800mL,
200mL methanol constant volumes are added to 1000mL.
Confining liquid：5g skimmed milk powers are weighed, 1 × TBS is added in and is settled to 100mL.
1×TBST：30.2g Tris salt is weighed, 8.766g sodium chloride after addition deionized water is settled to 1000mL, then adds
Enter 500 μ L Tween-20 mixings.
Primary antibody (secondary antibody) reaction solution：Primary antibody (secondary antibody) is diluted to reaction solution using 1 × TBST in proportion.
Fig. 1, pvdf membrane use the chemoluminescence method detection result figure after antibody A
M is protein molecular weight standard；1-9 swimming lanes for the mouse different disposal hepatic tissue of extraction holoprotein, on albumen
Sample amount is 50 μ g, the secondary antibody (Goat- marked for the first time with the HRP of primary antibody A (1: 1000 dilution factor) and 1: 5000 dilution factor
Anti-Rabbit after) reacting, the result of chemoluminescence method detection is used；
Fig. 2, pvdf membrane reuse the inspection of β-ACTIN antibody after washing away antibody using efficient protein matter Western blotting membrane regeneration liquor
Survey design sketch
M is protein molecular weight standard；1-9 swimming lanes for the mouse different disposal hepatic tissue of extraction holoprotein, on albumen
Sample amount is 50 μ g, the secondary antibody (Goat- marked for the first time with the HRP of primary antibody A (1: 1000 dilution) and 1: 5000 dilution factor
Anti-Mouse after) reacting, Western blotting is handled using efficient protein matter Western blotting membrane regeneration liquor after chemoluminescence method detection
After film, second of secondary antibody marked with the HRP of 1: 1000 diluted mouse anti-β-ACTIN and 1: 5000 dilution factor
(Goat-anti-Mouse) after secondary antibody reaction, the result of chemoluminescence method detection is used；
Fig. 3, pvdf membrane respectively using TUBULIN primary antibodies reaction (Fig. 3 A) and TUBULIN detection after reuse β-
The design sketch (Fig. 3 B) that ACTIN primary antibodies are detected and detected using substrate development process
Fig. 3 A, M are protein molecular weight standard；1-4 is the holoprotein of the different tissues of mice of extraction, and albumen applied sample amount is equal
For 50 μ g, the secondary antibody (Goat-anti- marked for the first time with primary antibody TUBULIN (1: 1000 dilution factor) and alkaline phosphatase (AP)
Rabbit) after (1: 1500 dilution factor) reaction, the result that is detected using substrate development process；
After Fig. 3 B, the Western blotting film for Fig. 3 A are handled using efficient protein matter Western blotting membrane regeneration liquor, second
With the secondary antibody (Goat- of the alkaline phosphatase (AP) of 1: 1000 diluted mouse anti-β-ACTIN and 1: 1500 dilution factor mark
Anti-Mouse after secondary antibody reaction), the result that is detected using substrate development process；
Fig. 4, NC film reuse the detection of β-ACTIN primary antibodies using antibody B reactions (Fig. 4 A) and after detecting simultaneously respectively
The design sketch (Fig. 4 B) detected using chemical method
Fig. 4 A, M are protein molecular weight standard；1-4 is the holoprotein of the different tissues of mice of extraction, and albumen applied sample amount is equal
For 50 μ g, the secondary antibody (Goat-anti- marked for the first time with antibody B (1: 1000 dilution factor) and horseradish peroxidase (HRP)
Rabbit) after (1: 5000 dilution factor) reaction, the result of chemoluminescence method detection is used；
After Fig. 4 B, the Western blotting film for Fig. 4 A are handled using efficient protein matter Western blotting membrane regeneration liquor, second
With the secondary antibody of the horseradish peroxidase (HRP) of 1: 1000 diluted mouse anti-β-ACTIN and 1: 5000 dilution factor mark
(Goat-anti-Mouse) after secondary antibody reaction, the result of chemoluminescence method detection is used；
Fig. 5, NC film reuse β-ACTIN antibody tests after being reacted respectively using antibody C reactions (Fig. 5 A) and antibody C to be made
The design sketch (Fig. 5 B) detected with substrate development process
Fig. 5 A, M are protein molecular weight standard；1-4 be extraction different disposal mouse lung tissue holoprotein, albumen
Applied sample amount is 50 μ g, the secondary antibody (Goat- marked for the first time with primary antibody C (1: 1000 dilution factor) and alkaline phosphatase (AP)
Anti-Mouse) after (1: 1500 dilution factor) reaction, the result that is detected using substrate development process；
After Fig. 5 B, the Western blotting film for Fig. 6 A are handled using efficient protein matter Western blotting membrane regeneration liquor, second
With (the Goat-anti- of the alkaline phosphatase (AP) of 1: 1000 diluted mouse anti-β-ACTIN and 1: 1500 dilution factor mark
Mouse after secondary antibody reaction), the result that is detected using substrate development process；
Fig. 6, pvdf membrane reuse β-ACTIN primary antibodies using antibody B reactions (Fig. 6 A) and after detecting respectively and make
The design sketch (Fig. 6 B) directly detected after reaction with the secondary antibody of fluorescent marker
Fig. 6 A, M are protein molecular weight standard：1-4 is the holoprotein of the different tissues of mice of extraction, and albumen applied sample amount is equal
For 50 μ g, the secondary antibody (Goat-anti- marked for the first time with antibody B (1: 1000 dilution factor) and horseradish peroxidase (HRP)
Rabbit) after (1: 5000 dilution factor) reaction, the result of chemoluminescence method detection is used；
After Fig. 6 B, the Western blotting film for Fig. 6 A are handled using efficient protein matter Western blotting membrane regeneration liquor, second
With the secondary antibody (Goat-anti- of the Dylight800 of 1: 1000 diluted mouse anti-β-ACTIN and 1: 8000 dilution factor marks
Mouse after secondary antibody reaction), the result that is directly detected using Image-forming instrument；
Specific embodiment
Embodiment 1, which handles pvdf membrane using efficient protein matter Western blotting membrane regeneration liquor and combines chemoluminescence method detection, to be exempted from
The result of epidemic disease blotting membrane
1st, experimental method
(1) murine liver tissue according to 100mg animal tissues add in 1 milliliter ratio add in general-purpose highly effective gross protein carry
Take reagent and homogenized；
(2) it is incubated 45 minutes on ice；
(3) 4 DEG C, 13000rpm is centrifuged 30 minutes；
(4) supernatant collected is animal total protein extract, can carry out subsequent experimental operation.
(5) protein solution that 24 μ L is taken to extract, add in 6 μ L 5 × loadingbuffer be sufficiently mixed uniformly after,
100 DEG C of heating are boiled is placed as after room temperature 4 DEG C after five minutes, and 13000rpm is centrifuged 20 minutes and to draw supernatant spare.
(6) measure of protein concentration：The measure of protein concentration is carried out using Bradford methods.
(7) glass plate of the 1.0mm cleaned thickness is fixed on encapsulating frame.
(8) 10% resolving polyacrylamide gel mixed liquor is prepared, the composition that polyacrylamide coagulates separation gel mixed liquor is
30% acrylamide/methene acrylamide, 1.5MTria-Cl (pH8.8), 10%SDS, 10% ammonium persulfate, TEMED.Take one
Full dose is loaded onto in glass plate after mixing for quantitative above-mentioned solution or reagent, and is slowly added to deionized water as water shutoff layer,
30min is placed at room temperature waits separation gel solidification.
(9) institute's water shutoff layer is outwelled after gelling to be separated is solid, 2mL polyacrylamides concentration glue is added in, while is inserted in glue surface
Upper comb.
(10) after gelling to be concentrated is solid, comb is slowly pulled out, is loaded onto in loading hole, 80V voltages run electrophoresis, treat that sample exists
When the interface of concentration glue and separation gel pressure is into a line, voltage is adjusted to 150V.
(11) after electrophoresis, two pieces of glass plates, excision concentration glue are separated.
(12) transferring film：By the clip (black portions of clip are in bottom) of transferring film according to sponge-Whatman filter paper-
After glue-pvdf membrane-Whatman filter paper-sponges-transparent clips part clips, transferring film behaviour is carried out with BioRad wet types membrane-transferring device
Make, add in transferring film buffer solution (constant current：400mA) turn 2 hours；
(13) close：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, when reaction 2 is small at room temperature；
(14) primary antibody is incubated：Add in primary antibody (antibody A)：Dilution ratio is 1: 1000, when room temperature reaction 2 is small；
(15) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(16) secondary antibody is incubated：Add in secondary antibody (HRP-Goat-anti-Rabbit), dilution ratio 1: 5000, room temperature reaction
When 1-2 is small；
(17) film is washed：TBST is washed 5 times, each 5min.
(18) develop the color：The colour developing that Western blotting film is carried out using chemical substrate luminescence reagent box is observed.
(19) Western blotting film regenerates：Efficient protein matter Western blotting membrane regeneration liquor is added in after secondary antibody reaction to react 20 minutes
It is washed 2 times, every time 5 minutes using 1 × TBS afterwards；
(20) it is re-closed：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, and it is small to react 2 at room temperature
When；
(21) primary antibody is incubated：It is 1: 1000 to add in primary antibody (β-ACTIN) dilution ratio, when room temperature reaction 2 is small；
(22) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(23) secondary antibody is incubated：Add in secondary antibody (HRP-Goat-anti-Mouse), dilution ratio 1: 5000, room temperature reaction
When 1-2 is small；
(24) film is washed：TBST is washed 5 times, each 5min.
(25) develop the color：The aobvious of Western blotting film is carried out using chemical substrate luminescence reagent box and using Las500 Image-forming instruments
Color is observed.
2nd, experimental result
Experimental result is shown, after first time purpose of usage protein antibodies A carries out protein immunoblotting detection, the results show
Antibody A does not have significantly specific (Fig. 1) identity of antigen.Add in efficient protein matter Western blotting membrane regeneration liquor reaction 20
It is washed 2 times using 1 × TBS after minute, 5 minutes every time, after removal antibody A is to the combination of antigen, then carries out second of β-ACTIN
The immune-blotting method of target protein, it is strong to carry out secondary β-ACTIN detection signal after the results show removal antibody A, background
Signal-to-noise ratio is very low (Fig. 2).Thus illustrate, efficient protein matter Western blotting membrane regeneration liquor can efficiently remove non-specific antibody and
The combination of antigen does not influence second of immune-blotting method, and second of detection result is good.
Embodiment 2 is exempted from using efficient protein matter Western blotting membrane regeneration liquor processing pvdf membrane and the detection of bound substrates development process
The result of epidemic disease blotting membrane
1st, experimental method
(1) murine liver tissue according to 100mg animal tissues add in 1 milliliter ratio add in general-purpose highly effective gross protein carry
Take reagent and homogenized；
(2) it is incubated 45 minutes on ice；
(3) 4 DEG C, 13000rpm is centrifuged 30 minutes；
(4) supernatant collected is animal total protein extract, can carry out subsequent experimental operation.
(5) protein solution that 24 μ L is taken to extract, add in 6 μ L 5 × loading buffer be sufficiently mixed uniformly after,
100 DEG C of heating are boiled is placed as after room temperature 4 DEG C after five minutes, and 13000rpm is centrifuged 20 minutes and to draw supernatant spare.
(6) measure of protein concentration：The measure of protein concentration is carried out using Bradford methods.
(7) glass plate of the 1.0mm cleaned thickness is fixed on encapsulating frame.
(8) 10% resolving polyacrylamide gel mixed liquor is prepared, the composition that polyacrylamide coagulates separation gel mixed liquor is
30% acrylamide/methene acrylamide, 1.5M Tris-Cl (pH8.8), 10%SDS, 10% ammonium persulfate, TEMED.Take one
Full dose is loaded onto in glass plate after mixing for quantitative above-mentioned solution or reagent, and is slowly added to deionized water as water shutoff layer,
30min is placed at room temperature waits separation gel solidification.
(9) institute's water shutoff layer is outwelled after gelling to be separated is solid, 2mL polyacrylamides concentration glue is added in, while is inserted in glue surface
Upper comb.
(10) after gelling to be concentrated is solid, comb is slowly pulled out, is loaded onto in loading hole, 80V voltages run electrophoresis, treat that sample exists
When the interface of concentration glue and separation gel pressure is into a line, voltage is adjusted to 150V.
(11) after electrophoresis, two pieces of glass plates, excision concentration glue are separated.
(12) transferring film：By the clip (black portions of clip are in bottom) of transferring film according to sponge-Whatman filter paper-
After glue-pvdf membrane-Whatman filter paper-sponges-transparent clips part clips, transferring film behaviour is carried out with Bio-Rad wet types membrane-transferring device
Make, add in transferring film buffer solution (constant current：400mA) turn 2 hours；
(13) close：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, when reaction 2 is small at room temperature；
(14) primary antibody is incubated：Add in primary antibody (Anti-TUBULIN)：Dilution ratio is 1: 1000, when room temperature reaction 2 is small；
(15) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(16) secondary antibody is incubated：The secondary antibody (AP-Goat-anti-Rabbit) of alkali phosphatase enzyme mark is added in, dilution ratio is
1: 1500, when room temperature reaction 1-2 is small；
(17) film is washed：TBST is washed 5 times, each 5min.
(18) develop the color：The colour developing that Western blotting film is carried out using alkaline phosphatase substrate NBT/BCIP kits is observed.
(19) Western blotting film regenerates：Efficient protein matter Western blotting membrane regeneration liquor is added in after secondary antibody reaction to react 20 minutes
It is washed 2 times, every time 5 minutes using 1 × TBS afterwards；
(20) it is re-closed：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, and it is small to react 2 at room temperature
When；
(21) primary antibody is incubated：It is 1: 1000 to add in primary antibody (β-ACTIN) dilution ratio, when room temperature reaction 2 is small；
(22) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(23) secondary antibody is incubated：Add in the secondary antibody (AP-Goat-anti-Mouse) of alkali phosphatase enzyme mark, dilution ratio 1
: 1500, when room temperature reaction 1-2 is small；
(24) film is washed：TBST is washed 5 times, each 5min.
(25) develop the color：The colour developing that Western blotting film is carried out using alkaline phosphatase substrate NBT/BCIP kits is observed.
2nd, experimental result
Experimental result shows that the antibody of first time purpose of usage albumen TUBULIN carries out protein immunoblotting detection simultaneously
After carrying out chromogenic reaction using substrate development process, the antibody of TUBULIN is good to the recognition effect of antigen (Fig. 3 A).After detection,
It adds in the reaction of efficient protein matter Western blotting membrane regeneration liquor to wash 2 times using 1 × TBS after twenty minutes, 5 minutes every time, removal was anti-
After the combination of original antibody, then carry out the immune-blotting method of second of β-ACTIN target protein, the results show β-ACTIN detections
Signal is clear and intensity is big (Fig. 3 B), thus illustrates that efficient protein matter Western blotting membrane regeneration liquor is equally applicable to substrate development process
The protein immunoblot detection of progress.
Embodiment 3 is immunized using efficient protein matter Western blotting membrane regeneration liquor processing NC films and combination chemoluminescence method detection
The result of blotting membrane
Experimental method
(1) murine liver tissue according to 100mg animal tissues add in 1 milliliter ratio add in general-purpose highly effective gross protein carry
Take reagent and homogenized；
(2) it is incubated 45 minutes on ice；
(3) 4 DEG C, 13000rpm is centrifuged 30 minutes；
(4) supernatant collected is animal total protein extract, can carry out subsequent experimental operation.
(5) protein solution that 24 μ L is taken to extract, add in 6 μ L 5 × loading buffer be sufficiently mixed uniformly after,
100 DEG C of heating are boiled is placed as after room temperature 4 DEG C after five minutes, and 13000rpm is centrifuged 20 minutes and to draw supernatant spare.
(6) measure of protein concentration：The measure of protein concentration is carried out using Bradford methods.
(7) glass plate of the 1.0mm cleaned thickness is fixed on encapsulating frame.
(8) 10% resolving polyacrylamide gel mixed liquor is prepared, the composition that polyacrylamide coagulates separation gel mixed liquor is
30% acrylamide/methene acrylamide, 1.5M Tris-Cl (pH8.8), 10%SDS, 10% ammonium persulfate, TEMED.Take one
Full dose is loaded onto in glass plate after mixing for quantitative above-mentioned solution or reagent, and is slowly added to deionized water as water shutoff layer,
30min is placed at room temperature waits separation gel solidification.
(9) institute's water shutoff layer is outwelled after gelling to be separated is solid, 2mL polyacrylamides concentration glue is added in, while is inserted in glue surface
Upper comb.
(10) after gelling to be concentrated is solid, comb is slowly pulled out, is loaded onto in loading hole, 80V voltages run electrophoresis, treat that sample exists
When the interface of concentration glue and separation gel pressure is into a line, voltage is adjusted to 150V.
(11) after electrophoresis, two pieces of glass plates, excision concentration glue are separated.
(12) transferring film：By the clip (black portions of clip are in bottom) of transferring film according to sponge-Whatman filter paper-
After glue-NC films-Whatman filter paper-sponges-transparent clips part clips, transferring film behaviour is carried out with Bio-Rad wet types membrane-transferring device
Make, add in transferring film buffer solution (constant current：400mA) turn 2 hours；
(13) close：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, when reaction 2 is small at room temperature；
(14) primary antibody is incubated：Add in primary antibody (antibody B)：Dilution ratio is 1: 1000, when room temperature reaction 2 is small；
(15) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(16) secondary antibody is incubated：Add in secondary antibody (HRP-Goat-anti-Rabbit), dilution ratio 1: 5000, room temperature reaction
When 1-2 is small；
(17) film is washed：TBST is washed 5 times, each 5min.
(18) develop the color：The colour developing that Western blotting film is carried out using chemical substrate luminescence reagent box is observed.
(19) Western blotting film regenerates：Efficient protein matter Western blotting membrane regeneration liquor is added in after secondary antibody reaction to react 20 minutes
It is washed 2 times, every time 5 minutes using 1 × TBS afterwards；
(20) close：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, when reaction 2 is small at room temperature；
(21) primary antibody is incubated：It is 1: 1000 to add in β-ACTIN dilution ratios, when room temperature reaction 2 is small；
(22) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(23) secondary antibody is incubated：Add in secondary antibody (HRP-Goat-anti-Mouse), dilution ratio 1: 5000, room temperature reaction
When 1-2 is small；
(24) film is washed：TBST is washed 5 times, each 5min.
(25) develop the color：The colour developing that Western blotting film is carried out using chemical substrate luminescence reagent box and Las500 Image-forming instruments is seen
It examines.
2nd, experimental result
Experimental result is shown, by rear first time purpose of usage protein antibodies on protein delivery to nitrocellulose filter (NC films)
After B carries out protein immunoblotting detection, the results show antibody B has significantly specific (Fig. 4 A) identity of antigen.Add
Enter the reaction of efficient protein matter Western blotting membrane regeneration liquor to wash 2 times using 1 × TBS after twenty minutes, 5 minutes every time, remove antibody B
After the combination of antigen, then the immune-blotting method of second of β-ACTIN target protein is carried out, after the results show removal antibody B
Carry out that secondary β-ACTIN detection signal is strong, and background signal-to-noise ratio is very low (Fig. 4 B), this result illustrates efficient protein matter Diagnosis of Sghistosomiasis
Mark membrane regeneration liquor can equally be well applied to the immune-blotting method again of NC films.
Embodiment 4 is immunized using efficient protein matter Western blotting membrane regeneration liquor processing NC films and the detection of bound substrates development process
The result of blotting membrane
Experimental method
(1) murine liver tissue according to 100mg animal tissues add in 1 milliliter ratio add in general-purpose highly effective gross protein carry
Take reagent and homogenized；
(2) it is incubated 45 minutes on ice；
(3) 4 DEG C, 13000rpm is centrifuged 30 minutes；
(4) supernatant collected is animal total protein extract, can carry out subsequent experimental operation.
(5) protein solution that 24 μ L is taken to extract, add in 6 μ L 5 × loading buffer be sufficiently mixed uniformly after,
100 DEG C of heating are boiled is placed as after room temperature 4 DEG C after five minutes, and 13000rpm is centrifuged 20 minutes and to draw supernatant spare.
(6) measure of protein concentration：The measure of protein concentration is carried out using Bradford methods.
(7) glass plate of the 1.0mm cleaned thickness is fixed on encapsulating frame.
(8) 10% resolving polyacrylamide gel mixed liquor is prepared, the composition that polyacrylamide coagulates separation gel mixed liquor is
30% acrylamide/methene acrylamide, 1.5M Tris-Cl (pH8.8), 10%SDS, 10% ammonium persulfate, TEMED.Take one
Full dose is loaded onto in glass plate after mixing for quantitative above-mentioned solution or reagent, and is slowly added to deionized water as water shutoff layer,
30min is placed at room temperature waits separation gel solidification.
(9) institute's water shutoff layer is outwelled after gelling to be separated is solid, 2mL polyacrylamides concentration glue is added in, while is inserted in glue surface
Upper comb.
(10) after gelling to be concentrated is solid, comb is slowly pulled out, is loaded onto in loading hole, 80V voltages run electrophoresis, treat that sample exists
When the interface of concentration glue and separation gel pressure is into a line, voltage is adjusted to 150V.
(11) after electrophoresis, two pieces of glass plates, excision concentration glue are separated.
(12) transferring film：By the clip (black portions of clip are in bottom) of transferring film according to sponge-Whatman filter paper-
After glue-NC films-Whatman filter paper-sponges-transparent clips part clips, transferring film behaviour is carried out with Bio-Rad wet types membrane-transferring device
Make, add in transferring film buffer solution (constant current：400mA) turn 2 hours；
(13) close：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, when reaction 2 is small at room temperature；
(14) primary antibody is incubated：Add in primary antibody C, dilution ratio 1: 1000, when room temperature reaction 2 is small；
(15) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(16) secondary antibody is incubated：Add in secondary antibody (AP-Goat-anti-Rabbit), dilution ratio 1: 1500, room temperature reaction
When 1-2 is small；
(17) film is washed：TBST is washed 5 times, each 5min.
(18) develop the color：The colour developing that Western blotting film is carried out using alkaline phosphatase substrate kit NBT/BCIP is observed.
(19) Western blotting film regenerates：Efficient protein matter Western blotting membrane regeneration liquor is added in after secondary antibody reaction to react 20 minutes
It is washed 2 times, every time 5 minutes using 1 × TBS afterwards；
(20) it is re-closed：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, and it is small to react 2 at room temperature
When；
(21) primary antibody is incubated：Add in primary antibody β-ACTIN, dilution ratio 1: 1000, when room temperature reaction 2 is small；
(22) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(23) secondary antibody is incubated：Add in the secondary antibody (AP-Goat-anti-Mouse) of alkali phosphatase enzyme mark, dilution ratio 1
: 1500, when room temperature reaction 1-2 is small；
(24) film is washed：TBST is washed 5 times, each 5min.
(25) develop the color：The colour developing that Western blotting film is carried out using alkaline phosphatase substrate kit NBT/BCIP is observed.
Experimental result
Experimental result is shown, egg is carried out using antibody C for the first time by rear on protein delivery to nitrocellulose filter (NC films)
After white matter immune-blotting method and use substrate development process carry out chromogenic reaction, the results show primary antibody C is to the recognition effect of antigen
Good (Fig. 5 A).After detecting for the first time, add in the reaction of efficient protein matter Western blotting membrane regeneration liquor and use 1 × TBS after twenty minutes
Washing 2 times, 5 minutes every time, after the combination for removing antigen-antibody, then carries out the Western blotting of second of β-ACTIN target protein
Detection, the results show β-ACTIN detection signals are strong (Fig. 5 B), illustrate that efficient protein matter Western blotting membrane regeneration liquor is equally applicable to
The protein immunoblot detection that substrate development process on NC films carries out.
Embodiment 5 directly detects immune using efficient protein matter Western blotting membrane regeneration liquor processing pvdf membrane and with reference to instrument
The result of blotting membrane
1st, experimental method
(1) murine liver tissue according to 100mg animal tissues add in 1 milliliter ratio add in general-purpose highly effective gross protein carry
Take reagent and homogenized；
(2) it is incubated 45 minutes on ice；
(3) 4 DEG C, 13000rpm is centrifuged 30 minutes；
(4) supernatant collected is animal total protein extract, can carry out subsequent experimental operation.
(5) protein solution that 24 μ L is taken to extract, add in 6 μ L 5 × loading buffer be sufficiently mixed uniformly after,
100 DEG C of heating are boiled is placed as after room temperature 4 DEG C after five minutes, and 13000rpm is centrifuged 20 minutes and to draw supernatant spare.
(6) measure of protein concentration：The measure of protein concentration is carried out using Bradford methods.
(7) glass plate of the 1.0mm cleaned thickness is fixed on encapsulating frame.
(8) 10% resolving polyacrylamide gel mixed liquor is prepared, the composition that polyacrylamide coagulates separation gel mixed liquor is
30% acrylamide/methene acrylamide, 1.5M Tris-Cl (pH8.8), 10%SDS, 10% ammonium persulfate, TEMED.Take one
Full dose is loaded onto in glass plate after mixing for quantitative above-mentioned solution or reagent, and is slowly added to deionized water as water shutoff layer,
30min is placed at room temperature waits separation gel solidification.
(9) institute's water shutoff layer is outwelled after gelling to be separated is solid, 2mL polyacrylamides concentration glue is added in, while is inserted in glue surface
Upper comb.
(10) after gelling to be concentrated is solid, comb is slowly pulled out, is loaded onto in loading hole, 80V voltages run electrophoresis, treat that sample exists
When the interface of concentration glue and separation gel pressure is into a line, voltage is adjusted to 150V.
(11) after electrophoresis, two pieces of glass plates, excision concentration glue are separated.
(12) transferring film：By the clip (black portions of clip are in bottom) of transferring film according to sponge-Whatman filter paper-
After glue-pvdf membrane-Whatman filter paper-sponges-transparent clips part clips, transferring film behaviour is carried out with BioRad wet types membrane-transferring device
Make, add in transferring film buffer solution (constant current：400mA) turn 2 hours；
(13) close：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, when reaction 2 is small at room temperature；
(14) primary antibody is incubated：Add in primary antibody (antibody D)：Dilution ratio is 1: 1000, when room temperature reaction 2 is small；
(15) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(16) secondary antibody is incubated：Add in secondary antibody (HRP-Goat-anti-Rabbit), dilution ratio 1: 5000, room temperature reaction
When 1-2 is small；
(17) film is washed：TBST is washed 5 times, each 5min.
(18) develop the color：The colour developing that Western blotting film is carried out using chemical substrate luminescence reagent box is observed.
(19) Western blotting film regenerates：Efficient protein matter Western blotting membrane regeneration liquor is added in after secondary antibody reaction to react 20 minutes
It is washed 2 times, every time 5 minutes using 1 × TBS afterwards；
(20) it is re-closed：Confining liquid (is configured) using TBS with 5% skimmed milk power to be closed, and it is small to react 2 at room temperature
When；
(21) primary antibody is incubated：It is 1: 1000 to add in primary antibody (β-ACTIN) dilution ratio, when room temperature reaction 2 is small；
(22) film is washed：TBST (TBS+0.1%Tween 20) is washed 5 times, each 5min；
(23) secondary antibody is incubated：Add in the secondary antibody (Dylight800-Goat-anti-Mouse) of fluorescent marker, dilution ratio
For 1: 8000, when room temperature reaction 1-2 is small；
(24) film is washed：TBST is washed 5 times, each 5min.
(25) develop the color：The colour developing observation of Western blotting film is directly carried out using azure Image-forming instruments.
2nd, experimental result
Experimental result shows that the antibody D of first time purpose of usage albumen carries out protein immunoblotting detection and use bottom
After object development process carries out chromogenic reaction, antibody D (Fig. 6 A) good to the recognition effect of antigen.After detection, efficient protein matter is added in
Western blotting membrane regeneration liquor reacts uses 1 × TBS washings 2 times after twenty minutes, 5 minutes every time, after the combination for removing antigen-antibody,
The immune-blotting method of second of β-ACTIN target protein is carried out again, and the results show β-ACTIN detection signals are clear and intensity is big
(Fig. 6 B) thus illustrates that the direct albumen for the secondary antibody that efficient protein matter Western blotting membrane regeneration liquor is equally applicable to fluorescent marker is exempted from
The detection of epidemic disease blotting membrane.
Claims (2)
1. application of a kind of protein immunoblotting membrane regeneration liquor in protein immunoblotting detection, which is characterized in that described
A kind of protein immunoblotting membrane regeneration liquor be made of 1~3M guanidine hydrochlorides, 2~5%SDS, 1~10%Tween20.
2. a kind of protein immunoblotting membrane regeneration liquor described in accordance with the claim 1 answering in protein immunoblotting detection
With, which is characterized in that a kind of protein immunoblotting membrane regeneration liquor is suitable for solid support nitre existing for protein
Acid cellulose film（NC films）And PVDF membrane（Pvdf membrane）.
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| CN111965371B (en) * | 2020-08-24 | 2023-11-24 | 上海雅酶生物医药科技有限公司 | Formula of protein blotting membrane antibody stripping solution and preparation and use methods thereof |
| CN112730847A (en) * | 2020-12-22 | 2021-04-30 | 哈尔滨赛信生物科技开发有限公司 | Kit for immunoblotting experiment by using antibody and application thereof |
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| A solution for stripping antibodies from polyvinylidene fluoride immunoblots for multiple reprobing;Yee-GuideYeung , E. RichardStanley;《Analytical Biochemistry》;20090319;第389卷(第1期);摘要，89页，Fig 2及其图注，91页左栏 * |
| The erasable Western blot;Scott H.Kaufmann et al;《Analytical Biochemistry》;19870215;第161卷(第1期);摘要 * |
| 影响 PVDF膜对蛋白吸附效率的主要因素;袁玉琦等;《氨基酸和生物资源》;20151010;第37卷(第3期);摘要 * |
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