阅读说明：本技术 一种蛋白质免疫印迹膜重生液及其使用方法 (A kind of protein immunoblotting film is lived again liquid and its application method ) 是由 张密 吴宇宁 于 2019-05-22 设计创作，主要内容包括：本发明了提供了一种可以快速高效地去除蛋白质免疫印迹膜上结合的一抗和二抗,蛋白质免疫印迹膜重生液及其使用方法。并且在不需要重新进行电泳、转膜,不破坏膜上的转印的蛋白质的情况下,使蛋白免疫印迹膜可以重复使用。(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.)

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.

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.