CN102692505A - Improved co-immunoprecipitation technical method - Google Patents

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

An improved co-immunoprecipitation technique

technical field

The invention belongs to the field of immunoassay, and in particular relates to an optimized co-immunoprecipitation technique. Based on the original co-immunoprecipitation technology, the present invention combines the co-immunoprecipitation technology with the protein cross-linking technology. That is, the protein cross-linking agent is used to cross-link the antigen-antibody complex, and after elution with a specific buffer, the western blot detection significantly reduces the contamination of the antibody heavy chain and light chain in the experiment, and increases the specificity of the method. The present invention improves the specificity of the co-immunoprecipitation method, has the characteristics of stability and repeatability, improves the experimental efficiency, and provides for in-depth research on molecular biology, molecular oncology, cell biology, biochemistry and other disciplines. helpful help.

Background technique

With the advent of the post-genome era, biomedical research has evolved from the hypothesis of one gene and one protein to the analysis of the functions of many genes or proteins at the omics level. Due to the complexity of various life activities of cells, the sequence information of the whole genome cannot interpret the biological functions of cells. The final product of genes, protein, is the ultimate executor of cell composition and function. Each protein does not independently complete the assigned function in the cell. In the complex structure of the cell and the highly ordered pathway network, it usually interacts with other proteins to form a large complex, and the interaction between proteins It is an important way for proteins to exert biological functions. Currently, scientists use large-scale proteomics techniques to develop and map extensive networks of protein-protein interactions in cells. So far, a variety of technical methods for studying protein interactions have been developed, including yeast two-hybrid system, phage display technology, GST pull-down technology, co-immunoprecipitation technology and tandem affinity purification combined with mass spectrometry analysis technology. The study of protein interaction and proteomics has laid a solid foundation.

Co-Immunoprecipitation (Co-Immunoprecipitation, IP) is a classic technique for studying protein interactions based on the specific binding between antibodies and antigens. The intact cells are mainly lysed with non-denaturing agents, which maintains the interaction between many proteins in the cell and facilitates the detection of the interaction between proteins. The antibody against protein A forms an immune complex with A and precipitates, while the protein B stably combined with A in the cell is simultaneously precipitated. Precipitation of protein B is based on physical interaction with A, a technique known as co-immunoprecipitation. This technology uses antigens and antibodies to form protein complexes to precipitate, dissolve the protein complexes, and then separate and purify the protein complexes through antibody affinity chromatography, and then use two-dimensional electrophoresis. The isolated protein is identified by techniques such as SDS-PAGE or mass spectrometry. The method is relatively stable, simple and widely used in the scientific research of cell biology, molecular biology and proteomics. Co-immunoprecipitation technology is an effective method to determine the interaction of two proteins under cell physiological conditions.

However, this classic technique has shortcomings in the actual operation process, that is, SDS-PAGE identifies the target protein and antibody in the isolated and purified protein. Since the mercaptoethanol contained in the loading buffer will cause the destruction of the disulfide bond between the heavy chain and the light chain of the antibody, the antibody molecule will become a heavy chain molecule (55KD) and a light chain molecule (25KD). Therefore, in addition to the target protein, heavy and light chain molecules can also be detected in the chromogenic reaction of western blot. Usually the amount of antibody used for immunoprecipitation is very large (1 μg), so when the size of the target protein is close to the heavy chain or light chain molecule, the Western blot signal of the heavy chain or light chain molecule is often due to the heavy chain or light chain signal being too strong. Affect the detection results of the target protein.

In order to effectively avoid the influence of the heavy chain and light chain in the experiment, it is necessary to improve the co-immunoprecipitation method to provide the specificity of the co-immunoprecipitation.

Contents of the invention

In order to achieve this improvement, the applicant compared and studied various technologies, and finally found that the combination of immunoprecipitation technology and protein cross-linking technology, that is, the use of protein cross-linking agents to cross-link antibodies and protein G (protein G) to form The complex, which is eluted with a specific buffer and detected by immunoblotting, can significantly reduce the contamination of antibody heavy and light chains in the experiment, thereby improving the specificity of co-immunoprecipitation. Furthermore, the present invention has been accomplished.

Specifically, in order to fully demonstrate the difference between the improved IP of the present invention and the traditional IP, the applicant is targeting protein binding known in the art, including the body apoptosis-inducing factor (AIF) and actin (actin) receptor interacting protein (RIP3) was compared with enolase (ENO1) in parallel, and it was found that the improved IP experiment of the present invention can significantly reduce heavy chain and light chain pollution, significantly improve the effect of co-immunoprecipitation, and overcome the false negative in the previous technology Or a false positive result.

Further, the present invention provides the following improved co-immunoprecipitation method, which comprises the following steps:

(1) Preparation materials:

(a) providing a sample to be tested containing a binary complex bound by protein X-Y;

(b) providing an antibody against protein X;

(c) providing protein G immobilized on microbeads, wherein said immobilization is covalently attached immobilization;

(d) providing Disuccinimidyl Suberate (DSS), Glycine and Tris reagents;

(2) Contact the antibody of (b) above with the protein G of (c), and physically combine the protein G immobilized on the microbeads with the Fc fragment of the anti-X antibody under appropriate conditions to obtain microbeads-protein G - a tribody complex of anti-X antibodies,

(3) Treat the tribody complex obtained in the above (2) with disuccinimide suberate (DSS) to carry out appropriate covalent cross-linking to form the tribody complex of microbeads-protein G-anti-X antibody. body complex cross-linked product,

(4) contacting the tribody complex cross-linked product obtained in (3) above with the dibody complex in (a) under suitable conditions to form a microbead-protein G-anti-X antibody-X-Y five-body complex; The pentameric complex was obtained by centrifugation,

(5) Heat and denature the five-body complex obtained in (4) above, and centrifuge to obtain the precipitate of the three-body complex cross-linked product containing microbeads-protein G-anti-X antibody, and the supernatant containing protein X and protein Y. clear,

(6) SDS electrophoresis was performed on the supernatant, and routine western blot analysis (Western blot) was performed.

On the other hand, in the above-mentioned improved co-immunoprecipitation method provided by the present invention,

The microbeads in step (1) can be agarose microbeads, magnetic beads, or non-magnetic particles;

The sample to be tested in step (1) can be cell lysate, tissue fluid, body fluid, blood, protein complex, etc., and can be tissue fluid containing a target protein of 40-60kD size, 20-30kD size, 55kD or 25kD size, Body fluids, blood, protein complexes, etc.

The protein G immobilized on microbeads in step (1) can be purchased commercially, or can be covalently immobilized through the technical manual.

An appropriate washing step is optionally added in steps (1)-(6), wherein the washing buffer can be a buffer commonly used in the art such as PBS and cell lysate.

Appropriate conditions in step (2) refer to conventional conditions such as buffer, pH, temperature, etc., which can be determined by those skilled in the art based on common knowledge and technical manuals.

The covalent cross-linking of step (3) can use a variety of commonly used cross-linking agents and cross-linking conditions in the art, such as the cross-linking system composed of disuccinimide suberate (DSS), the cross-linking in the present invention The conditions include that the concentration of the DSS cross-linking agent is 5Mm, the PBS buffer system is used, the cross-linking temperature is room temperature, and the cross-linking time can be 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes minutes, 110 minutes and 120 minutes, preferably 40-120 minutes, more preferably 60-90 minutes.

Embodiments and effects of the present invention will be further described below in conjunction with the accompanying drawings.

Description of drawings

Figure 1: Schematic comparing classical and modified immunoprecipitation techniques.

Figure 2: Using the interaction between AIF and actin to compare the imprinting results of the traditional IP method and the improved IP method of the present invention.

Figure 3: Results of determining optimal antibody and protein G optimal crosslinking times.

Figure 4: Blot results for discovering the interaction of RIP3 protein with ENO1 using the improved IP technique.

In order to clearly illustrate the present invention, the applicant cites the following examples, wherein the examples are not used to limit the present invention, but are only used to illustrate the present invention.

Example 1: Comparison between the improved co-immunoprecipitation method and the traditional IP method in identifying the interaction between AIF and actin

The steps are as follows:

Extraction of total intracellular protein:

1.1 Use a petri dish with a diameter of 10 cm to culture the esophageal cancer cell line KYSE140 cells (a human esophageal cancer cell line from Japan, donated by Professor Shimada Y, Kyoto University), in the PRM1640 medium containing 10% fetal bovine serum, Cultivate for 3 days, and when the confluence of the cells reaches 90%, wash the cells with 4°C pre-cooled 1×PBS, 5ml/time×2 times. After washing, add pre-cooled 0.6ml cell lysate to the cell culture dish, the ingredients include 50mM Tris, pH7.4, 150mM sodium chloride, 1% triton-100 (membrane permeabilizer), 0.1 %SDS, and quickly scrape the cells with a cell scraper and collect them in a 15ml centrifuge tube. Sonication on ice (sonication for 10 seconds, with an interval of 4 seconds, a total of 10 times).

1.2 Divide the sonicated protein lysate in 1.1 into two 1.5ml centrifuge tubes, and centrifuge at 4°C at 12,000 rpm for 15 minutes. The supernatant was collected, and the protein was quantified by Bradford method.

1.3 According to the quantitative results in 1.2, divide the protein lysate at 1mg/ml into 2ml cryovials and store on ice. For co-immunoprecipitation, take 1-2 mg of total protein cell lysate for experiments.

1. Binding and cross-linking of antibody Ab to protein G microbeads

1.1 Pipette 600 μl of PBS, add anti-AIF antibody (Santa Cruz Company) and mix well. Protein G microbeads were added, and protein G was ordered from a commercial company (GE, USA). Rotate and incubate at 4°C for 2 hours; centrifuge at low speed at 4°C for 5 minutes, discard the supernatant, and leave the precipitate to obtain the tribody complex of microbeads-protein G-anti-AIF antibody;

1.2 Add PBS to the precipitate in 2.1 for washing, centrifuge at low speed at 4°C for 5 minutes x 2 times, and discard the supernatant. Divide into two parts, one of which is the control, except that the following 2.3 and 2.4 are not performed, the rest of the operations are the same;

1.3 Add 100 μl of 5mM DSS cross-linking buffer to the tribody complex of microbeads-protein G-anti-AIF antibody obtained in 2.2. The main components include 5mM DSS, 0.02M PBS, pH=7.4, and incubate at room temperature for 1 hour; then Add 100μl 0.5M Tris (PH=7.4) to terminate the remaining DSS cross-linking agent, incubate with rotation for 20 minutes, centrifuge at 4°C for 5 minutes at low speed, discard the supernatant, and keep the precipitate to obtain microbeads -Protein G-anti-AIF antibody trimer complex cross-link; control without this step.

1.4 Add 1ml of protein-free cell lysate pre-cooled at 4°C to the cross-linked trimer complex obtained in 2.3 above for washing, centrifuge at low speed at 4°C for 5 minutes x 1 time, remove the supernatant, and add 40 μl of The protein-free cell lysate was pre-cooled at 4°C to obtain the cross-linked microbead-protein G-anti-AIF antibody trimer complex, and stored at 4°C for future use.

2. Immunoprecipitation (IP)

2.1 Use 1ml of protein solution containing KYSE140 whole cell lysis pre-cooled at 4°C, take 0.5ml of protein solution containing whole cell lysis and add it to the control tube A in 2.1 above, and add another 0.5ml of protein solution of whole cell lysis to 2.4 Incubate B in a 1.5ml centrifuge tube containing the trimer complex cross-linked product, rotate and incubate at 4°C for 2 hours; centrifuge at low speed at 4°C for 5 minutes, discard the supernatant, and leave the precipitate to obtain microbeads-protein G-anti-AIF antibody - pentameric complex of antigen X-Y;

2.2 Add 1ml of protein-free cell lysate pre-cooled at 4°C to the control tube A and microbeads-protein G-anti-AIF antibody-antigen X-Y five-body complex tube B obtained in 3.1 above for washing. For the unbound protein on the complex, centrifuge at 4°C for 5 minutes at low speed three times, discard the supernatant and leave the precipitate.

2.3 Add 50ul 1xSDS loading buffer to the control tube A obtained in the above 3.2 and microbeads-protein G-anti-AIF antibody-antigen XY five-body complex tube B respectively, and its components include 0.12M Tris, 5% glycerol, 0.4% SDS, 1% β-mercaptoethanol and 0.02% bromophenol blue, denatured in boiling water at 100°C for 10 minutes, centrifuged at 10000g×5min, and collected the supernatant.

2.4 SDS electrophoresis, and western blot detection.

Samples on SDS electrophoresis include KYSE140 whole cell lysed protein solution, supernatant obtained from classic IP experiment, loading antibody, supernatant obtained from improved IP experiment, and eluted supernatant from IP experiment. After SDS electrophoresis, anti-AIF Antibody, anti-Actin antibody and anti-PARP antibody were routinely detected by western blot.

The result of embodiment 1:

The total protein of the esophageal cancer cell line KYSE140 cells was subjected to the classic co-immunoprecipitation of anti-AIF antibody as a control, and after the improved co-immunoprecipitation of the protein cross-linking step, the detection results are shown in Figure 2, in which lanes 1 and 2 are The result of the classic IP experiment, there are a lot of heavy and light chain contaminations in the lanes, as indicated by the white arrows; Lane 3 is the loading antibody (Santa Cruz Company) as a positive control for the light and heavy chains of the antibody; 4 and 5 Lane 1 is the result of the improved IP experiment. It can be clearly seen that after cross-linking with the protein cross-linking agent, the contamination of the light chain and heavy chain of the antibody is effectively eliminated; Lane 6 and 7 are the eluted supernatant of the IP experiment.

The results of AIF immunoblotting showed that a specific band was displayed at a molecular weight of 67kDa, indicating that the triple complex of microbeads-protein G-anti-AIF antibody effectively recognized the AIF antigen, immunoaffinity precipitated the AIF antigen, and the immunoblotting was effective of detected AIF protein molecules.

The results of Western blotting of Actin showed that Actin could interact with AIF protein, and the cross-linking agent did not affect the interaction between Actin and AIF, which was consistent with the results reported in the literature that AIF could interact with Actin protein[ K, et al., 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. Fu Yurong Screening and Identification of Interacting Proteins of Truncated Mitochondrial Apoptosis-Inducing Factor Biology, Chemistry and Biophysics Advances 2009, 36(1):42-48].

Poly ADP-ribose polymerase (PARP) is a protein localized in the nucleus. The results of literature survey showed that there was no report of any interaction between PARP and AIF. Therefore, we used the samples collected above to conduct SDS electrophoresis and immunological analysis of PARP. The western blot results showed that no interaction between PARP and AIF was found in the classical IP experiments (lanes 1 and 2) and the improved IP experiments (4 and 5). It shows that both the classic IP experiment and the improved IP experiment can effectively detect the protein interaction.

Example 2: Determining Optimal Antibody and Protein G Optimal Crosslinking Time

All materials and methods are the same as in Example 1, wherein only the crosslinking time has been changed, the incubation time of step 2.3 in Example 1 is changed to: 50min, 60min and 90min, and the results are shown in the swimming lane 3- of Fig. 2 5.

In step 3.4, the samples on SDS electrophoresis include the protein solution of KYSE140 whole cell lysis, the supernatant obtained from the classic IP experiment, the cross-linked 50min, 60min and 90min supernatant obtained from the improved IP experiment, and the eluted supernatant of the IP experiment After SDS electrophoresis and transmembrane transfer, conventional immunoblotting was performed using anti-AIF antibody, anti-Actin antibody and anti-heat shock protein 60 (HSP60) antibody.

The result of embodiment 2:

The total protein of the esophageal cancer cell line KYSE140 cells was cross-linked at three time points of 30min, 60min and 90min after the classic co-immunoprecipitation of anti-AIF antibody as a control, and the co-immunoprecipitation of the improved protein cross-linking step, as shown in Figure 3 TL is the sample of total cell protein, and lane 1 is the result of classic IP experiment, there are a lot of heavy and light chain contamination of the antibody in the lane, as indicated by the arrow; lane 2 is the loaded antibody as the light chain and heavy chain of the antibody positive control; lanes 3, 4, and 5 are improved IP experiments, and protein cross-linking agents are cross-linked at different time points for 50 min, 60 min, and 90 min, which effectively eliminates the contamination of antibody light and heavy chains; 6, 7, and Lane 8 is the elution supernatant of the IP experiment, which are the first, second and third elutions respectively. According to the above-mentioned improved co-immunoprecipitation method of the present invention, by changing the cross-linking time, as the cross-linking time prolongs, the contamination of antibody light chain and heavy chain is effectively eliminated, and the optimal cross-linking time point is determined.

The results of AIF immunoblotting showed that a specific band was displayed at a molecular weight of 67kDa, indicating that the triple complex of microbeads-protein G-anti-AIF antibody effectively recognized the AIF antigen, immunoaffinity precipitated the AIF antigen, and the immunoblotting was effective of detected AIF protein molecules.

The results of Western blotting of Actin showed that Actin could interact with AIF protein, and the cross-linking agent did not affect the interaction between Actin and AIF, which was consistent with the results reported in the literature that AIF could interact with Actin protein.

Heat shock protein 60 (HSP60) is a 60kDa protein highly expressed in cells, accounting for 5%-10% of the total protein in cells. Under stress conditions, such as heat shock, the expression of heat shock proteins is significantly increased, which can protect cells. Heat shock proteins act as "molecular chaperones" that can bind to other proteins and assist in protein translocation, folding and assembly. Heat shock proteins have played an important role in various diseases [Azem, A et al., Characterization of a functional GroEL-14(GroES-7)-2 chaperonin hetero-oligomer.Science 265:653-656, 1994.Schmidt, M et al. , Symmetric complexes of GroE chaperonins as part of the functional cycle. Science 265: 656-659, 1994.]. The results of the literature survey showed that there was no report of any interaction between HSP60 and AIF. Therefore, HSP60 was selected as a control for non-specific binding to AIF. We used the samples collected above to detect by SDS electrophoresis and conventional Western blotting. The Western blotting of HSP60 The results showed that there was no interaction between HSP60 and AIF after classical IP experiments (lane 1) and improved IP experiments (3, 4 and 5). It shows that both the classic IP experiment and the improved IP experiment can truly detect the interacting proteins in the cell and avoid non-specific binding proteins.

Example 3: Using the improved co-immunoprecipitation technique to identify the interaction between RIP3 protein and ENO1 The steps are as follows:

1. Extraction of total intracellular protein:

1.1 Use two petri dishes with a diameter of 10 cm to culture the cells of the esophageal cancer cell line KYSE140, a human esophageal cancer cell line from Japan, in the PRM1640 medium containing 10% fetal bovine serum, culture for 1 day, and wait for the fusion of the cells up to 80% degree. One plate of cells was treated with 10uM anticancer drug cisplatin for 24 hours (B), and the other plate was not treated as a control (A). Wash the cells with 4°C pre-cooled 1×PBS, 5ml/time×2 times. After washing, add pre-cooled 0.6ml cell lysate to the cell culture dish, the ingredients include 50mM Tris, pH7.4, 150mM sodium chloride, 1% triton-100 (membrane permeabilizer), 0.1 %SDS, and quickly scrape the cells with a cell scraper and collect them into a 1.5ml centrifuge tube, and sonicate on ice (sonication for 10 seconds with a gap of 4 seconds, 10 times in total).

1.2 Divide the sonicated protein lysate in 1.1 into two 1.5ml centrifuge tubes, and centrifuge at 4°C at 12,000 rpm for 15 minutes. The supernatant was collected, and the protein was quantified by Bradford method.

1.3 According to the quantitative results in 1.2, divide the protein lysate at 1mg/ml into 2ml cryovials and store on ice. For co-immunoprecipitation, take 1-2 mg of total protein cell lysate for experiments.

2. Binding and cross-linking of antibody Ab to protein G microbeads

2.1 Pipette 600 μl of PBS, add anti-RIP3 antibody (product of ABCAM Company) and mix well. Protein G microbeads were added, and protein G was ordered from a commercial company (GE, USA). Rotate and incubate at 4°C for 2 hours; centrifuge at low speed at 4°C for 5 minutes, discard the supernatant, and leave the precipitate to obtain the tribody complex of microbeads-protein G-anti-RIP3 antibody;

2.2 Add PBS to the precipitate in 2.1 for washing, centrifuge at low speed at 4°C for 5 minutes x 2 times, discard the supernatant;

2.3 Add 100 μl of 5mM DSS cross-linking buffer to the tribody complex of microbeads-protein G-anti-RIP3 antibody obtained in 2.2. The main components include 5mM DSS, 0.02M PBS, pH=7.4, and incubate at room temperature for 1 hour; Then add 100μl 0.5M Tris(PH=7.4) to terminate the remaining DSS cross-linking agent, incubate with rotation for 20 minutes, centrifuge at 4°C for 5 minutes at low speed, discard the supernatant, and keep the precipitate to obtain micro Glob-Protein G-anti-RIP3 antibody trimer complex cross-link; control without this step.

2.4 Add 1ml of protein-free cell lysate pre-cooled at 4°C to the cross-linked trimer complex obtained in 2.3 above for washing, centrifuge at low speed at 4°C for 5 minutes x 1 time, remove the supernatant, and add 40 μl of The protein-free cell lysate was pre-cooled at 4°C to obtain the cross-linked microbead-protein G-anti-RIP3 antibody trimer complex, and stored at 4°C for later use.

3. Immunoprecipitation (IP)

3.1 Use 1ml of the protein solution containing KYSE140 whole cell lysis pre-cooled at 4°C (A) and the protein solution of KYSE140 whole cell lysis treated with cisplatin (B), respectively take 0.5ml of the protein solution containing whole cell lysis and add it to 2.4. Incubate in a 1.5ml centrifuge tube of the tribody complex cross-linked product at 4°C for 2 hours; centrifuge at 4°C at low speed for 5 minutes, discard the supernatant, and leave the precipitate to obtain microbeads-protein G-anti-RIP3 antibody-antigen X-Y five-body complex;

3.2 Add 1ml of protein-free cell lysate pre-cooled at 4°C to the tube of microbeads-protein G-anti-RIP3 antibody-antigen X-Y five-body complex obtained in 3.1 above to wash the unbound complex. Centrifuge at low speed for 5 minutes at 4°C for 3 times, discard the supernatant and leave the precipitate.

3.3 Add 50ul 1xSDS loading buffer to the microbeads-protein G-anti-RIP3 antibody-antigen X-Y five-body complex tube B obtained in 3.2 above. The components include 0.12M tris, 5% glycerol, Denature 0.4% SDS, 1% β-mercaptoethanol and 0.02% bromophenol blue in boiling water at 100°C for 10 minutes, centrifuge at 10000g×5min, and collect the supernatant.

3.4 SDS electrophoresis, and Western blot detection.

The samples on SDS electrophoresis include the whole cell lysed protein solution containing A and B, and the supernatants obtained from the improved IP experiment including A and B respectively. After SDS electrophoresis, anti-RIP3 antibody and anti-enolase (ENO1) antibody were used Routine immunoblotting was performed.

The test result of embodiment 3

The total protein of esophageal cancer cell line KYSE140 cells (A) and the protein solution of KYSE140 whole cell lysate treated with chemotherapy drug cisplatin (B), after the co-immunoprecipitation of anti-RIP3 antibody to improve the protein cross-linking step, conventional western blot detection , as shown in Figure 4, TL is the sample of total cell protein, lane 1 is the total protein of esophageal cancer cell line KYSE140 cells; lane 2 is the protein solution of KYSE140 whole cells lysed by the chemotherapeutic drug cisplatin; lane 3 Lane 1 is the result of the improved IP experiment of A; Lane 4 is the result of the improved IP experiment of B, which effectively eliminates the contamination of the light chain and heavy chain of the antibody. According to the above-mentioned improved co-immunoprecipitation method of the present invention, the contamination of antibody light chain and heavy chain is effectively eliminated, and the specificity of the experiment is increased.

The results of immunoblotting of RIP3 showed a specific band at a molecular weight of 57kDa, indicating that the triple complex of microbeads-protein G-anti-RIP3 antibody effectively recognized the RIP3 antigen, immunoaffinity precipitated the RIP3 antigen, and the immunoblotting was effective of detected AIF protein molecules.

The results of immunoblotting against enolase ENO1 showed that after the co-immunoprecipitation of the protein cross-linking step improved by the anti-RIP3 antibody, a specific band with a molecular weight of 47 kDa was displayed, which was the band of the ENO1 protein molecule. It suggested that ENO1 could interact with RIP3 protein, but the cross-linker did not affect the interaction between ENO1 and RIP2. Using this improved IP experimental technique, we discovered the interaction between ENO1 and RIP3 protein.

In a word, the above-mentioned multiple examples prove that the improved IP method of the present invention can significantly reduce the influence of heavy and light chains on immunoassay, and can be used for immunoassay detection of various proteins. At the same time, the cross-linking step is very simple and the effect is very obvious.

Claims (8)

1. An improved co-immunoprecipitation method, comprising the steps of: (1) Preparation materials: (a) providing a sample to be tested containing a binary complex bound by protein X-Y; (b) providing an antibody against protein X; (c) providing protein G immobilized on microbeads, wherein said immobilization is covalently attached immobilization; (2) Contact the antibody of (b) above with the protein G of (c), and physically combine the protein G immobilized on the microbeads with the Fc fragment of the anti-X antibody under appropriate conditions to obtain microbeads-protein G - a tribody complex of anti-X antibodies, (3) Appropriately covalently cross-linking the trimer complex obtained in (2) above to provide a trimer complex cross-linked product of microbeads-protein G-anti-X antibody, (4) contacting the tribody complex cross-linked product obtained in (3) above with the dibody complex in (a) under suitable conditions to form a microbead-protein G-anti-X antibody-X-Y five-body complex; The pentameric complex was obtained by centrifugation, (5) Heat and denature the five-body complex obtained in (4) above, and centrifuge to obtain the precipitate of the three-body complex cross-linked product containing microbeads-protein G-anti-X antibody, and the supernatant containing protein X and protein Y. clear, (6) SDS electrophoresis was performed on the supernatant, and immunoassay of conventional co-immunoprecipitation was performed. 2. The improved co-immunoprecipitation method of claim 1, wherein the microbeads in step (1) are agarose microbeads, magnetic beads, or non-magnetic particles. 3. The improved co-immunoprecipitation method of claim 1, wherein the test sample in step (1) is a test sample containing 40-60kD size, 20-30kD size, 55kD or 25kD size target protein. 4. The improved co-immunoprecipitation method of claim 1, wherein the test sample in step (1) is a cell lysate selected from cell lysate, tissue fluid, body fluid, blood and protein complexes. 5. The improved co-immunoprecipitation method according to claim 1, wherein the covalent cross-linking in step (3) can use various cross-linking agents and cross-linking conditions commonly used in the art. 6. The improved co-immunoprecipitation method of claim 1, wherein the cross-linking agent in step (3) is disuccinimide suberate. 7. The improved co-immunoprecipitation method of claim 1, wherein the cross-linking temperature in step (3) is room temperature. 8. The improved co-immunoprecipitation method of claim 1, wherein the cross-linking time of step (3) is selected from 40-120 minutes, 50-100 minutes and 60-90 minutes.

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