CN102692505A - Improved co-immunoprecipitation technical method - Google Patents
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Abstract
The invention belongs to an immunodetection field and specifically relates to an optimized co-immunoprecipitation technology. Based on an original co-immunoprecipitation technology, the co-immunoprecipitation technology is combined with a protein crosslink technology. That is to say, a protein crosslinking agent is used to crosslink an antigen antibody complex; elution is carried out by the use of a specific buffer; and the immunoblotting detection remarkably reduces the pollution of heavy chain and light chain of the antibody in the experiment. According to the invention, specificity of the co-immunoprecipitation method is raised, the method is characterized by stability and repeatability, experiment efficiency is improved, and the method provides beneficial assistance for in-depth research in molecular biology, molecular oncology, cell biology, biochemistry and the like.
Description
Technical field
The invention belongs to field of immunodetection, be specifically related to a kind of co-immunoprecipitation technology of optimization.The present invention is technical at original co-immunoprecipitation, and the co-immunoprecipitation technology is combined with the protein-crosslinking technology.Promptly utilize the crosslinked antigen antibody complex of protein-crosslinking agent, through special buffer solution elution, Western blotting detects the pollution significantly reduced heavy chain of antibody and light chain in the experiment, has increased the specificity of method.The present invention has improved the specificity of co-immunoprecipitation method; Have stability and repeatable characteristics; Improved conventional efficient, be the deep molecular biology that carries out, the research of subjects such as molecular weight tumor, cell biology, biological chemistry provides useful help.
Background technology
Along with the arriving of genome times afterwards comprehensively (post genomie era), biomedical research has been deduced from the hypothesis of an albumen of a gene and has been learned the function of numerous genes of horizontal analysis or albumen in group.Because the various vital movement phenomenon numerous and complicated of cell, complete genomic sequence information can not be annotated the biological function of cell.And the ultimate product albumen of gene is only the final executor of cellularity and function.Each albumen is not independently in cell, to accomplish the function of being given; In the path network of cell labyrinth and high-sequential; Common and other protein interaction forms big compound, and the interaction between the protein-protein is the important channel of protein performance biological function.Current, scientist utilizes large-scale proteomic techniques development and has drawn interactional network collection of illustrative plates between the intracellular protein widely.Developed the technical method of multiple research protein interaction so far; Comprise yeast two-hybrid system, display technique of bacteriophage, GST settling test (GST pull-down) technology, co-immunoprecipitation is technological and tandem affinity purification associating analytical technique of mass spectrum, for solid foundation has been established in the research of protein interaction and proteomics.
(Co-Immunoprecipitation IP) is the classical technology that is used to study protein interaction that is combined into the basis with specificity between antibody and the antigen to co-immunoprecipitation.Mainly utilize non-denaturant cracking intact cell, kept the interaction between numerous protein in the cell, be convenient to detect the interaction between the protein.Utilize antibody and the A of anti-protein A to form immune complex and deposition, and the interior PROTEIN B with the A stable bond of cell is precipitated simultaneously.The deposition of PROTEIN B is based on the physical property interaction with A, and this technology is called as co-immunoprecipitation.This techniques make use antigen-antibody forms the albumen composition deposition, with the protein complex dissolving, through the affinity chromatography of antagonist the albumen composition separation and purification is come out, and then passes through two dimensional electrophoresis.Technical appraisement such as SDS-PAGE or mass spectrum separates the albumen that obtains.Its method is relatively stable, method is easy and be widely used in the scientific research of cell biology, molecular biology and proteomics, and the co-immunoprecipitation technology is to confirm two kinds of protein interactional effective ways under the cell physiological condition.
But the technology of this classics exists not enough in the operating process of reality, and promptly SDS-PAGE identifies in the albumen of separation and purification, contains destination protein and antibody.Can cause the heavy chain of antibody and the disulfide bond between the light chain to destroy owing to contain mercaptoethanol in the sample loading buffer, thereby make antibody molecule become heavy chain molecule (55KD) and light chain molecule (25KD).Therefore, in the Western blotting chromogenic reaction,, can also detect heavy chain and light chain molecule except detecting the destination protein.The antibody amount that is generally used for immunoprecipitation is big (1 μ g) very; So when the size of destination protein is divided the period of the day from 11 p.m. to 1 a.m near heavy chain or light chain, the Western blotting signal of heavy chain or light chain molecule usually influences the testing result of destination protein owing to heavy chain or light chain overflow.
For the influence that heavy chain in effectively avoiding testing and light chain cause experiment, necessary the co-immunoprecipitation method is improved, the specificity of co-immunoprecipitation is provided.
Summary of the invention
In order to realize this improvement; The applicant compares multiple technologies and studies, and finds at last the co-immunoprecipitation technology to be combined the compound that promptly utilizes protein-crosslinking agent cross-linking antibody and Protein G (protein G) to form with the protein-crosslinking technology; Through special buffer solution elution; Western blotting detects, and can significantly reduce the pollution of heavy chain of antibody and light chain in the experiment, has improved the specificity of co-immunoprecipitation thus.And then accomplished the present invention.
Particularly; In order fully to show the difference of improvement IP of the present invention and Traditional IP; The applicant is to protein combination well known in the art; Comprise that body apoptosis inducing factor (AIF) has carried out parallel comparison with actin (actin) receptor interacting protein (RIP3) with enolase (ENO1); The result finds that the improved IP of the present invention experiment can significantly alleviating heavy chain pollutes with light chain, and the effect of co-immunoprecipitation is significantly improved, and has overcome in the past in the technology perhaps false-positive result of false negative.
Further, the invention provides following improved co-immunoprecipitation method, it comprises the steps:
(1) prepare material:
(a) testing sample that contains the binary compound that albumin X-Y combines is provided;
(b) antibody of anti-albumin X is provided;
(c) Protein G that is fixed on the microballon is provided, wherein said fixedly is covalently bound fixing;
(d) double amber imide suberate (DSS), glycocoll and trishydroxymethylaminomethane reagent are provided;
(2) antibody of above-mentioned (b) and the Protein G of (c) are contacted, the Protein G that is fixed on the microballon are combined with the Fc fragment physical property of anti-X antibody, obtain the trisome complex of microballon-Protein G-anti-X antibody,
(3) the trisome complex and the double amber imide suberate (DSS) that above-mentioned (2) are obtained are handled, and carry out suitable covalent cross-linking, have formed the trisome complex cross-linking agent of microballon-Protein G-anti-X antibody,
(4) the trisome complex cross-linking agent that above-mentioned (3) is obtained contacts under appropriate condition with the binary compound in (a), forms microballon-Protein G-anti-X antibody-X-Y five body constituents compound; Through centrifugal this five body constituents compound that obtains,
(5) above-mentioned (4) are obtained the five body constituents compound and carry out heat denatured and handle, through centrifugal, obtain containing the trisome complex cross-linking agent of microballon-Protein G-anti-X antibody deposition, contain the supernatant of albumin X and Y albumen,
(6) supernatant is carried out the SDS electrophoresis, carry out routine immunization engram analysis (Western blot).
In addition on the one hand, in the above-mentioned improved co-immunoprecipitation method provided by the invention,
The microballon of step (1) can be the agarose microballon, has the magnetic bead of magnetic, or does not have the particle of magnetic;
The testing sample of step (1) can be a cell pyrolysis liquid, can be tissue fluid, body fluid, blood; Albumen compositions etc. can be to contain the 40-60kD size, 20-30kD size, the tissue fluid of the destination protein of 55kD or 25kD size; Body fluid, blood, albumen composition etc.
The Protein G that is fixed on the microballon in the step (1) can be commercial the purchase, also can carry out Covalent Immobilization through technical manual.
The suitable washing step of choosing wantonly in step (1)-(6) of adding, wherein lavation buffer solution can be this areas such as PBS and cell pyrolysis liquid damping fluids commonly used.
Appropraite condition in the step (2) is meant damping fluid, pH, and normal conditions such as temperature, those skilled in the art can confirm according to common practise and technical manual fully.
The covalent cross-linking of step (3) can use multiple crosslinking chemical commonly used in this area and crosslinked condition, the cross-linking system formed of double amber imide suberate (DSS) for example, and the crosslinked condition among the present invention comprises that the concentration of DSS crosslinking chemical is 5Mm, adopts the PBS buffer system; Crosslinked temperature is a room temperature, and crosslinking time can be 30 minutes, 40 minutes, and 50 minutes; 60 minutes, 70 minutes, 80 minutes, 90 minutes; 100 minutes, 110 minutes and 120 minutes, preferred 40-120 minute, more preferably 60-90 minute.
Below in conjunction with accompanying drawing embodiments of the invention and effect are described further.
Description of drawings
Fig. 1: the schematic diagram of relatively classical and more improved immunoprecipitation technology.
Fig. 2: the trace result who comes comparison Traditional IP method and the improved IP method of the present invention with the interaction of AIF and actin.
Fig. 3: the result who confirms the best crosslinking time of optimum antibody and Protein G.
Fig. 4: the interactional trace result who utilizes improved IP scientific discovery RIP3 albumen and ENO1.
In order clearly to set forth the present invention, the applicant has enumerated following embodiment, and wherein said example is not used for limiting the present invention, only is used for setting forth the present invention.
Embodiment 1: improved co-immunoprecipitation method and Traditional IP method are in the interactional comparison of identifying AIF and actin
Its step is following:
The extraction of total protein in the cell:
1.1 using the double dish cultured cell esophageal cancer cell of diameter 10cm is that the KYSE140 cell is (from a strain human esophageal carcinoma cell line of Japan; By Shimada Y professor, be so kind as to give the Kyoto University), in containing the PRM1640 nutrient culture media of 10% hyclone; Cultivated 3 days; The degrees of fusion of treating cell reaches 90% degree, uses 1 * PBS of 4 ℃ of precoolings to wash cell, 5ml/ time * 2 times.After the washing, in Tissue Culture Dish, add the 0.6ml cell pyrolysis liquid of precooling, ingredient comprises 50mM trishydroxymethylaminomethane pH7.4; 150mM sodium chloride; 1%triton-100 (pass through film), 0.1%SDS, and scrape cell harvesting rapidly with the cell sleaker and go in the centrifuge tube of 15ml.Ultrasonic on ice (ultrasonic 10 seconds, gap 4 seconds, totally 10 times).
1.2 the protein lysate behind ultrasonic in 1.1 is sub-packed in two 1.5ml centrifuge tubes, and 4 ℃ are centrifugal, and 12,000 rev/mins, 15 minutes.Collect supernatant, Bradford method protein quantification.
1.3 according to the quantitative result in 1.2, protein lysate divided according to 1mg/ml install in the frozen pipe of 2ml, preserves on ice.During co-immunoprecipitation, the cell pyrolysis liquid of getting the 1-2mg total protein experimentizes.
1. antibody A b and Protein G microballon combines with crosslinked
1.1 draw 600 μ l PBS, add antibody (the Santa Cruz company) mixing of anti-AIF.Add the Protein G microballon, Protein G is ordered (U.S. GE company) from commercial company.4 ℃ of rotations were hatched 2 hours; 4 ℃ of low-speed centrifugals 5 minutes, supernatant discarded stays deposition, promptly obtains microballon-Protein G-anti-AIF antibody ternary complex;
Wash 4 ℃ of low-speed centrifugals 5 minutes * 2 times, supernatant discarded 1.2 add PBS in the deposition in 2.1.Be divided into two parts, a copy of it is contrast, removes and does not carry out outside following 2.3,2.4, and all the other operations are all identical;
1.3 add the crosslinked damping fluid of 100 μ l5mM DSS in the microballon-Protein G that in 2.2, obtains-anti-AIF antibody ternary complex, its principal ingredient comprises 5mM DSS, 0.02M PBS, PH=7.4, and the room temperature rotation was hatched 1 hour; Add 100 μ l 0.5M trishydroxymethylaminomethanes (PH=7.4) then, be used to stop remaining DSS crosslinking chemical, rotation was hatched 20 minutes; 4 ℃ of low-speed centrifugals 5 minutes; Abandon supernatant, leave and take deposition, promptly obtain microballon-Protein G-anti-AIF antibody trisome complex cross-linking agent; This step is not carried out in contrast.
1.4 in the trisome complex cross-linking agent that above-mentioned 2.3 obtain; The not protein-contg cell pyrolysis liquid 1ml that adds 4 ℃ of precoolings washs; 4 ℃ of low-speed centrifugals 5 minutes * 1 time remove supernatant, add the not protein-contg cell pyrolysis liquid of 4 ℃ of precoolings of adding of 40 μ l; Promptly obtain microballon-Protein G-anti-AIF antibody trisome complex cross-linking agent, 4 ℃ of preservations are subsequent use.
2. immunoprecipitation (IP)
2.1 the protein liquid 1ml that contains the full lysis of KYSE140 with 4 ℃ of precoolings; Get the protein liquid that 0.5ml contains full lysis respectively and add A in the control tube in above-mentioned 2.1; The protein liquid of the full lysis of 0.5ml adds B in the 1.5ml centrifuge tube that contains trisome complex cross-linking agent in 2.4 in addition, and 4 ℃ of rotations were hatched 2 hours; 4 ℃ of low-speed centrifugals 5 minutes, supernatant discarded stays deposition, promptly obtains the five body constituents compound of microballon-Protein G-anti-AIF antibody-antigen X-Y;
2.2 in control tube A and microballon-Protein G-anti-AIF antibody-antigen X-Y five body constituents complex B pipe that above-mentioned 3.1 obtain; The not protein-contg cell pyrolysis liquid 1ml that adds 4 ℃ of precoolings respectively washs; In order to unconjugated albumen on the washing compound; 4 ℃ of low-speed centrifugals 5 minutes * 3 times, supernatant discarded stays deposition.
2.3 to above-mentioned 3.2 control tube that obtain
AAnd microballon-Protein G-anti-AIF antibody-antigen X-Y five body constituents complex
BIn the pipe, add 50ul 1xSDS sample-loading buffer respectively, its composition comprises 0.12M trishydroxymethylaminomethane, 5% glycerine, 0.4%SDS, 1% beta-mercaptoethanol and 0.02% bromophenol blue; Sex change is 10 minutes in 100 ℃ of boiling water; Centrifugal, 10000g * 5min collects supernatant.
2.4SDS electrophoresis carries out Western blotting and detects.
Sample comprises on the SDS electrophoresis; The supernatant that the protein liquid of the full lysis of KYSE140, classical IP experiment is obtained, go up an appearance antibody, improve the IP experiment supernatant that obtains, wash-out supernatant that IP tests; Through the SDS electrophoresis, adopt the conventional Western blotting of carrying out of anti-AIF antibody, anti-Actin antibody and anti-PARP antibody to detect.
The result of embodiment 1:
Esophageal cancer cell be the KYSE140 total protein of cell through the classical co-immunoprecipitation of anti-AIF antibody as contrast; With improved behind the co-immunoprecipitation of protein-crosslinking step; Having shown testing result like Fig. 2, wherein is classical IP result of experiment in the swimming lane 1,2; There is the pollution of lot of antibodies heavy chain and light chain in the swimming lane, like the white arrow indication; 3 roads are the positive control of last appearance antibody (Santa Cruz company) as light chain of antibody and heavy chain; 4 and 5 roads are improved IP experimental results, can see significantly, after the protein-crosslinking agent is crosslinked, have effectively eliminated the pollution of light chain of antibody and heavy chain; 6 and 7 roads are the wash-out supernatant of IP experiment.
The Western blotting result of AIF shows; At molecular weight is that the 67kDa place shows a specific band; Explain microballon-Protein G-anti-AIF antibody three complex effective recognition AIF antigen, immune affinity precipitation AIF antigen, Western blotting effectively detects the AIF protein molecular.
The Western blotting result of Actin shows; Actin can with the AIF protein-interacting; And crosslinking chemical does not influence the interaction of Actin and AIF; There is the AIF of report can consistent [
K with document with the result of Actin protein-interacting; Deng the people; Chemical cross-linking leads to two high molecular mass aggregates of rat alpha 1 beta 1 integrin differing in their conformation but not in their composition.FEBS Lett.1995,16; 373 (3): 234-8. pays beautiful honor etc., blocks the screening and the identification of organism of molded lines plastochondria apoptosis inducing factor interacting protein, chemistry and biophysics progress 2009,36 (1): 42-48].
Poly ADP ribose polymerase (PARP) is a kind of endonuclear albumen that is positioned; The literature survey result shows that PARP and AIF do not see any interactional report, and therefore, we use the sample of above-mentioned collection; Through the SDS electrophoresis; The Western blotting result of PARP shows, through classical IP experiment (swimming lane 1 and 2) and improved IP experiment (4 and 5), does not all see the interactional relation of PARP and AIF.Explain that classical IP experiment of above-mentioned process and improved IP experiment all can effectively detect the interaction of albumen.
Embodiment 2: confirm the best crosslinking time of optimum antibody and Protein G
All material is identical with embodiment 1 with method, wherein only aspect crosslinking time, changes, and the incubation time with step 2.3 among the embodiment 1 changes to respectively: 50min, 60min and 90min, its result is in shown in the swimming lane 3-5 of Fig. 2.
Sample comprises on the SDS electrophoresis in step 3.4; The wash-out supernatant of the supernatant that the protein liquid of the full lysis of KYSE140, classical IP experiment are obtained, the supernatant that improves IP experiment crosslinked 50min, 60min and the 90min of obtaining, IP experiment; Through the SDS electrophoresis; After changeing film, adopt the conventional Western blotting of carrying out of anti-AIF antibody, anti-Actin antibody and heat resistanceheat resistant shock protein 60 (HSP60) antibody to detect.
The result of embodiment 2:
Esophageal cancer cell be the KYSE140 total protein of cell through the classical co-immunoprecipitation of anti-AIF antibody as contrast; With the co-immunoprecipitation that improves the protein-crosslinking step, adopt 30min, 60min and three time points of 90min to carry out crosslinked, as shown in Figure 3; TL is an appearance on the total protein of cell; 1 road is classical IP result of experiment, has the pollution of lot of antibodies heavy chain and light chain in the swimming lane, like the arrow indication; 2 roads are the positive control of last appearance antibody as light chain of antibody and heavy chain; 3,4 and 5 roads are improved IP experiments, and it is crosslinked to carry out the protein-crosslinking agent through different time points 50min, 60min and 90min, has effectively eliminated the pollution of light chain of antibody and heavy chain; 6,7 and 8 roads are the wash-out supernatant of IP experiment, are respectively for the first time, for the second time with eluent for the third time.According to the improvement co-immunoprecipitation method of the invention described above, through changing crosslinking time,, effectively eliminated the pollution of light chain of antibody and heavy chain along with the prolongation of crosslinking time, confirm best crosslinking time point.
The Western blotting result of AIF shows; At molecular weight is that the 67kDa place shows a specific band; Explain microballon-Protein G-anti-AIF antibody three complex effective recognition AIF antigen, immune affinity precipitation AIF antigen, Western blotting effectively detects the AIF protein molecular.
The Western blotting result of Actin shows, Actin can with the AIF protein-interacting, and crosslinking chemical does not influence the interaction of Actin and AIF, has the AIF of report can be consistent with the result of Actin protein-interacting with document.
Heat shock protein 60 (HSP60) is a 60kDa, and high abundance expressed proteins in cell accounts for the 5%-10% of cell protein total amount.Coercing under the incentive condition, like heat shock, expression of heat shock obviously raises, and can play a protective role by pair cell.Heat shock protein plays " molecular chaperones ", can with other protein combination, assist the transposition of albumen, folding and assembling.Heat shock protein has been brought into play vital role [Azem in multiple disease; People such as A; Characterization of a functional GroEL-14 (GroES-7)-2chaperonin hetero-oligomer.Science 265:653-656,1994.Schmidt, people such as M; Symmetric complexes of GroE chaperonins as part of the functional cycle.Science 265:656-659,1994.].The literature survey result shows that HSP60 and AIF do not see any interactional report, therefore, selects the contrast of HSP60 conduct and the non-specific bond of AIF for use; We use the sample of above-mentioned collection; Through the SDS electrophoresis, detect through conventional Western blotting, the Western blotting result of HSP60 shows; Through classical IP experiment (swimming lane 1) and improved IP experiment (3,4 and 5), all do not see the interactional relation of HSP60 and AIF.Explain that classical IP experiment of above-mentioned process and improved IP experiment all can truly detect interactional albumen in the cell, have avoided non-specific bond albumen.
Embodiment 3: adopt its step of interaction of improved co-immunoprecipitation technical appraisement RIP3 albumen and ENO1 following:
1. the extraction of total protein in the cell:
1.1 using two double dish cultured cell esophageal cancer cells of diameter 10cm is the KYSE140 cell; Strain human esophageal carcinoma cell line from Japan; In containing the PRM1640 nutrient culture media of 10% hyclone, cultivated 1 day, treat that the degrees of fusion of cell reaches 80% degree.Add 10uM cancer therapy drug cisplatin treated 24 hours (B) in the one dish cell, another dish does not process as contrast (A).Use 1 * PBS of 4 ℃ of precoolings to wash cell, 5ml/ time * 2 times.After the washing, in Tissue Culture Dish, add the 0.6ml cell pyrolysis liquid of precooling, ingredient comprises 50mM trishydroxymethylaminomethane pH7.4; 150mM sodium chloride, 1%triton-100 (passing through film), 0.1%SDS; And scrape cell harvesting rapidly with the cell sleaker and go in the centrifuge tube of 1.5ml; Ultrasonic on ice (ultrasonic 10 seconds, gap 4 seconds, totally 10 times).
1.2 the protein lysate behind ultrasonic in 1.1 is sub-packed in two 1.5ml centrifuge tubes, and 4 ℃ are centrifugal, and 12,000 rev/mins, 15 minutes.Collect supernatant, Bradford method protein quantification.
1.3 according to the quantitative result in 1.2, protein lysate divided according to 1mg/ml install in the frozen pipe of 2ml, preserves on ice.During co-immunoprecipitation, the cell pyrolysis liquid of getting the 1-2mg total protein experimentizes.
2. antibody A b and Protein G microballon combines with crosslinked
2.1 draw 600 μ l PBS, add antibody (ABCAM Company products) mixing of anti-RIP3.Add the Protein G microballon, Protein G is ordered (U.S. GE company) from commercial company.4 ℃ of rotations were hatched 2 hours; 4 ℃ of low-speed centrifugals 5 minutes, supernatant discarded stays deposition, promptly obtains microballon-Protein G-anti-RIP3 antibody ternary complex;
2.3 add the crosslinked damping fluid of 100 μ l 5mM DSS in the microballon-Protein G that in 2.2, obtains-anti-RIP3 antibody ternary complex, its principal ingredient comprises 5mM DSS, 0.02M PBS, PH=7.4, and the room temperature rotation was hatched 1 hour; Add 100 μ l 0.5M trishydroxymethylaminomethanes (PH=7.4) then, be used to stop remaining DSS crosslinking chemical, rotation was hatched 20 minutes; 4 ℃ of low-speed centrifugals 5 minutes; Abandon supernatant, leave and take deposition, promptly obtain microballon-Protein G-anti-RIP3 antibody trisome complex cross-linking agent; This step is not carried out in contrast.
2.4 in the trisome complex cross-linking agent that above-mentioned 2.3 obtain; The not protein-contg cell pyrolysis liquid 1ml that adds 4 ℃ of precoolings washs; 4 ℃ of low-speed centrifugals 5 minutes * 1 time remove supernatant, add the not protein-contg cell pyrolysis liquid of 4 ℃ of precoolings of adding of 40 μ l; Promptly obtain microballon-Protein G-anti-RIP3 antibody trisome complex cross-linking agent, 4 ℃ of preservations are subsequent use.
3. immunoprecipitation (IP)
3.1 protein liquid (B) with the full lysis of KYSE140 of the protein liquid 1ml (A) that contains the full lysis of KYSE140 of 4 ℃ of precoolings and cisplatin treated; Get protein liquid that 0.5ml contains full lysis respectively and add in 2.4 and contain in the 1.5ml centrifuge tube of trisome complex cross-linking agent, 4 ℃ of rotations were hatched 2 hours; 4 ℃ of low-speed centrifugals 5 minutes, supernatant discarded stays deposition, promptly obtains the five body constituents compound of microballon-Protein G-anti-RIP3 antibody-antigen X-Y;
3.2 in microballon-Protein G-anti-RIP3 antibody-antigen X-Y five body constituents complex pipe that above-mentioned 3.1 obtain; The not protein-contg cell pyrolysis liquid 1ml that adds 4 ℃ of precoolings respectively washs; In order to unconjugated albumen on the washing compound; 4 ℃ of low-speed centrifugals 5 minutes * 3 times, supernatant discarded stays deposition.
3.3 in microballon-Protein G-anti-RIP3 antibody-antigen X-Y five body constituents complex B pipe that above-mentioned 3.2 obtain; Add 50ul 1xSDS sample-loading buffer respectively; Its composition comprises 0.12M trishydroxymethylaminomethane, 5% glycerine, 0.4%SDS, 1% beta-mercaptoethanol and 0.02% bromophenol blue, and sex change is 10 minutes in 100 ℃ of boiling water, and is centrifugal; 10000g * 5min collects supernatant.
3.4SDS electrophoresis carries out Western blotting and detects.
Sample comprises on the SDS electrophoresis, comprises the protein liquid of the full lysis of A and B, the supernatant of improvement IP experiment obtains to comprise respectively A and B, through the SDS electrophoresis, adopts the routine immunization trace that carries out of anti-RIP3 antibody, anti-enolase (ENO1) antibody to detect.
The test findings of embodiment 3
Esophageal cancer cell is a KYSE140 total protein of cell (A) and through the protein liquid (B) of the full lysis of the KYSE140 of chemotherapeutics cisplatin treated; After improving the co-immunoprecipitation of protein-crosslinking step through anti-RIP3 antibody; The routine immunization trace detects; As shown in Figure 4, TL is an appearance on the total protein of cell, and 1 road is that A is an oesophagus cancerous cell line KYSE140 total protein of cell; 2 roads are that B is promptly through the protein liquid of the full lysis of the KYSE140 of chemotherapeutics cisplatin treated; 3 roads are that A is through improved IP experimental result; 4 roads be B through improved IP experimental result, effectively eliminated the pollution of light chain of antibody and heavy chain.According to the improvement co-immunoprecipitation method of the invention described above, effectively eliminated the pollution of light chain of antibody and heavy chain, increased the specificity of experiment.
The Western blotting result of RIP3 shows; At molecular weight is that the 57kDa place shows a specific band; Explain microballon-Protein G-anti-RIP3 antibody three complex effective recognition RIP3 antigen, immune affinity precipitation RIP3 antigen, Western blotting effectively detects the AIF protein molecular.
The Western blotting result of anti-enolase ENO1 shows, behind the co-immunoprecipitation through anti-RIP3 antibody improvement protein-crosslinking step, is that the 47kDa place shows a specific band at molecular weight, is ENO1 protein molecular band.Prompting ENO1 can with the RIP3 protein-interacting, and crosslinking chemical does not influence the interaction of ENO1 and RIP2.Utilize this improved IP experimental technique, found ENO1 can with the interaction of RIP3 albumen.
Prove through above-mentioned a plurality of embodiment that in a word improved IP method of the present invention can significantly reduce the influence of heavy and light chain to immunoassay, the immunoassay that is used in multiple protein detects.Described cross-linking step is very easy simultaneously, and effect is very obvious.
Claims (8)
1. improved co-immunoprecipitation method, it comprises the steps:
(1) prepare material:
(a) testing sample that contains the binary compound that albumin X-Y combines is provided;
(b) antibody of anti-albumin X is provided;
(c) Protein G that is fixed on the microballon is provided, wherein said fixedly is covalently bound fixing;
(2) antibody of above-mentioned (b) and the Protein G of (c) are contacted, the Protein G that is fixed on the microballon are combined with the Fc fragment physical property of anti-X antibody, obtain the trisome complex of microballon-Protein G-anti-X antibody,
(3) the trisome complex that above-mentioned (2) is obtained carries out suitable covalent cross-linking to be handled, and the trisome complex cross-linking agent of microballon-Protein G-anti-X antibody is provided,
(4) the trisome complex cross-linking agent that above-mentioned (3) is obtained contacts under appropriate condition with the binary compound in (a), forms microballon-Protein G-anti-X antibody-X-Y five body constituents compound; Through centrifugal this five body constituents compound that obtains,
(5) above-mentioned (4) are obtained the five body constituents compound and carry out heat denatured and handle, through centrifugal, obtain containing the trisome complex cross-linking agent of microballon-Protein G-anti-X antibody deposition, contain the supernatant of albumin X and Y albumen,
(6) supernatant is carried out the SDS electrophoresis, carry out the immunoassay of routine immunization co-precipitation.
2. the improved co-immunoprecipitation method of claim 1, wherein, the microballon of step (1) is the agarose microballon, has the magnetic bead of magnetic, or do not have the particle of magnetic.
3. the improved co-immunoprecipitation method of claim 1, wherein, the testing sample of step (1) is to contain the 40-60kD size, 20-30kD size, the testing sample of 55kD or 25kD size destination protein.
4. the improved co-immunoprecipitation method of claim 1, wherein, the testing sample of step (1) is a cell pyrolysis liquid, is selected from cell pyrolysis liquid, tissue fluid, body fluid, blood and albumen composition.
5. the improved co-immunoprecipitation method of claim 1, wherein, the covalent cross-linking of step (3) can use multiple crosslinking chemical commonly used in this area and crosslinked condition.
6. the improved co-immunoprecipitation method of claim 1, wherein, the crosslinking chemical of step (3) is the double amber imide suberate.
7. the improved co-immunoprecipitation method of claim 1, wherein, the crosslinked temperature of step (3) is a room temperature.
8. the improved co-immunoprecipitation method of claim 1, wherein, the crosslinking time of step (3) is selected from 40-120 minute, 50-100 minute with 60-90 minute.
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| CN105938147A (en) * | 2016-05-10 | 2016-09-14 | 浙江大学 | Method for detecting proteins in serum by using combination of co-immunoprecipitation and Western blot technology |
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| CN112255061A (en) * | 2020-10-13 | 2021-01-22 | 南开大学 | Method for separating and detecting protein by immunoprecipitation |
| CN112824905A (en) * | 2019-11-20 | 2021-05-21 | 中国科学院大连化学物理研究所 | Method for detecting interaction or affinity between ligand and protein based on solvent-induced protein precipitation |
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| CN105938147A (en) * | 2016-05-10 | 2016-09-14 | 浙江大学 | Method for detecting proteins in serum by using combination of co-immunoprecipitation and Western blot technology |
| CN109164260A (en) * | 2018-08-20 | 2019-01-08 | 广州伯信生物科技有限公司 | A kind of co-immunoprecipitation method that can exclude antibody signal interference |
| CN112824905A (en) * | 2019-11-20 | 2021-05-21 | 中国科学院大连化学物理研究所 | Method for detecting interaction or affinity between ligand and protein based on solvent-induced protein precipitation |
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| CN112824905B (en) * | 2019-11-20 | 2022-06-07 | 中国科学院大连化学物理研究所 | Method for detecting interaction or affinity between ligand and protein based on solvent-induced protein precipitation |
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