CN113092760A - Method for detecting interaction between protein and protein - Google Patents

Method for detecting interaction between protein and protein Download PDF

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CN113092760A
CN113092760A CN202110268247.1A CN202110268247A CN113092760A CN 113092760 A CN113092760 A CN 113092760A CN 202110268247 A CN202110268247 A CN 202110268247A CN 113092760 A CN113092760 A CN 113092760A
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protein
buffer
centrifugal column
antibody
cell lysate
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王宏婷
王存琴
汪荣斌
陈颖
臧莉
黄超
蒋永旭
孙绪强
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Wannan Medical College
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract

The invention provides a method for detecting interaction between protein and protein, which comprises the following steps: step one, adding the antibody into a centrifugal column for solidification treatment; step two, agarose matrix pretreatment of cell lysate; step three, placing the antibody after the solidification treatment on agarose matrix pretreatment cell lysate for treatment; step four, performing co-immunoprecipitation and elution; and step five, performing SDS-PAGE analysis on the eluted flow-through liquid, and detecting the interaction between the target proteins. The method is simple and effective, has good specificity and extremely high accuracy. The experimental operation is further simplified, and the experimental time is shortened.

Description

Method for detecting interaction between protein and protein
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for detecting interaction between protein and protein.
Background
The interaction between protein and protein constitutes one main component of cell biochemical reaction network, and the protein-protein interaction network and transcription regulation network have important significance in regulating cell and its signal. At present, the commonly used method for detecting the interaction between protein and protein mainly comprises a yeast two-hybrid system, pull-down technology and the like. The principle of the yeast two-hybrid system is that after the target protein and the bait protein are specifically combined, the bait protein is combined with a promoter of the reporter gene to start the expression of the reporter gene in yeast cells, if an expression product of the reporter gene is detected, the interaction between the target protein and the reporter gene is indicated, otherwise, the interaction between the target protein and the reporter gene is not indicated. The Pull-down technique uses immobilized, labeled bait or tag proteins (biotin-, PolyHis-or GST-) to extract proteins from cell lysates that interact with them.
In the first method, the hybridization system analyzes that the interaction between proteins is localized in the nucleus, and many of the interactions between proteins depend on post-translational processes such as glycosylation, disulfide bond formation, etc., and these reactions cannot proceed in the nucleus. While the second method may have false positives. Perhaps due to adsorption from the influence of charge interactions rather than physiological interactions. Also, Pull-down is generally a validation of in vitro interactions and cannot be used to detect in vivo interactions at the cellular level.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art. Therefore, the invention provides a method for detecting protein-protein interaction, aiming at improving the purity and specificity of protein and accurately detecting the protein-protein interaction.
In view of the above object, the present invention provides a method for detecting protein-protein interaction, comprising the steps of:
step one, adding the antibody into a centrifugal column for solidification treatment;
step two, agarose matrix pretreatment of cell lysate;
step three, placing the antibody after the solidification treatment on agarose matrix pretreatment cell lysate for treatment;
step four, performing co-immunoprecipitation and elution;
and step five, performing SDS-PAGE analysis on the eluted flow-through liquid, and detecting the interaction between the target proteins.
The method for curing in the first step comprises the following steps:
a1, pretreating the first centrifugal column, and adding an HER2 antibody, a cross-linking buffer solution and ultrapure water into the first centrifugal column;
a2, incubating by a gyrometer at room temperature, centrifuging and reserving a first centrifugal column.
The solute of the crosslinking buffer in the step A1 is 0.01M Na3PO40.15M NaCl; the pH was 7.2.
The method for the cracking treatment in the second step comprises the following steps:
b1, washing the cells by a modified buffer solution, adding a precooled IP lysis/washing buffer solution, incubating on ice and centrifuging;
b2, adding agarose resin slurry into the second centrifugal column, centrifuging, adding a cross-linking buffer solution, and centrifuging again to obtain a second centrifugal column containing resin;
b3, adding the cell lysate into a second centrifugal column containing resin, and reserving flow-through liquid after incubation and centrifugation; wherein the mass-volume ratio of the cell lysate to the agarose resin slurry is 1:80 mg/muL.
The flow-through liquid obtained from B3 was added to the first spin column after A2 treatment, and the flow-through liquid was retained after centrifugation.
HCC1806 breast cancer cells were lysed in B1 using modified Du's PBS buffer and IP lysis/wash buffer.
The solute of the improved Du's PBS is 0.008M Na3PO4、0.002M K3PO40.14M NaCl, 0.01M KCl; the pH is 7; the solutes of the IP lysis/wash buffer were 0.025M Tris, 0.15M NaCl, 0.001M EDTA, 1% NP-40, 5% glycerol; the pH was 7.4.
The method for treating in the third step is to add the flow-through liquid obtained from B3 into the first centrifugal column after A2 treatment, and retain the flow-through liquid after centrifugation. And washing the treated first centrifugal column twice by using an IP lysis/washing buffer solution, centrifuging, then carrying out BCA protein concentration determination, detecting whether the flow-through liquid exists or not, and if the protein exists, continuing to wash.
In the first step, the mass of the added antibody is 10-75 mu g, and the volume of the solution is adjusted to 200 mu L. The buffer concentration was 1 ×.
In step one, the resin is activated by covalently coupling the antibody to an amine group. Avoids the phenomenon that the final eluent contains the heavy chain and the light chain of the antibody, and the band formed by the migration of the heavy chain and the light chain is possibly overlapped with the band of the target protein, thereby causing error of the experimental result.
In the fifth step, a non-reduced type lane mark loading buffer solution is adopted, wherein the solute comprises 0.3M Tris-HCl, 5% SDS and 50% glycerol, and the lane mark tracing dye; the pH was 6.8.
The invention has the beneficial effects that:
1. the invention separates natural protein complex from cell lysate by directly fixing purified antibody on agarose matrix. I.e. immunoprecipitating a specific antigen (bait protein) with an antibody and co-precipitating any proteins that interact with the antigen (target proteins). The method is simple and effective, has good specificity and extremely high accuracy. The experimental operation is further simplified, and the experimental time is shortened.
2. The high-efficiency centrifugal column system used by the invention further simplifies the experiment operation and shortens the experiment time. The centrifugal column system comprises a centrifugal column, a spiral cover, a bottom cover, a spiral cap and a gasket. When the volume is scaled up, a gasket may be inserted to facilitate washing. The screw cap has a flip-top that can be used during the washing step. The centrifugal column can be sealed by a screw cap during storage.
3. The resin can be regenerated and stored, so that the equipment consumed by experiments is greatly saved, and the experiment cost is saved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph comparing the interaction of EGFR with HER2 in breast cancer cells MDA-MB-468 measured using the method of the present invention with conventional co-Immunoprecipitation (IP) and Western Blot (WB).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to specific embodiments below.
It is to be noted that technical terms or scientific terms used in the embodiments of the present invention should have the ordinary meanings as understood by those having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined. The use of "first," "second," and similar terms in this disclosure is not intended to indicate any order, quantity, or importance, but rather is used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items.
Example 1
The coupling resin and reagents were equilibrated to room temperature.
The 20 Xcrosslinking buffer was diluted with ultrapure water to 1X, and 2ml of 1 Xcrosslinking buffer was prepared for each reaction, i.e., 100. mu.L of 20 Xcrosslinking buffer and 1.9ml of ultrapure water were taken to prepare 1 Xcrosslinking buffer.
The bottles of resin were lightly spin-coupled to obtain a uniform suspension. Using a large bore truncated tip, 20. mu.L of the resin slurry was added to the first spin column. The column was placed in a microcentrifuge tube, centrifuged at 1000 Xg for 1min and the flow-through liquid discarded.
The resin was washed with 200. mu.L of 1 Xcrosslinking buffer, centrifuged at 1000 Xg for 1min, and the flow-through liquid was discarded and the bottom cap inserted.
Mu.g of HER2 antibody was added, and the desired volume of antibody was 57.14. mu.L calculated from the antibody concentration, and 10. mu.L of 20 Xcrosslinking buffer and 180. mu.L of ultrapure water were added to make the final buffer concentration 1X.
In a fume hood, 3. mu.L of a 5mol/L solution of sodium cyanoborohydride was added to the reaction system.
And (3) covering a screw cap, and incubating on a vortex mixer for 120min at room temperature to ensure that the slurry is in a suspension state in the incubation process.
The bottom plug is detached and retained, and the screw cap is unscrewed. The first spin column was placed in a collection tube and centrifuged at 1000 Xg for 1 min. Flow through liquid was preserved to verify antibody binding efficiency.
The screw cap was removed, 200. mu.L of 1 Xcrosslinking buffer was added to the column, centrifuged at 1000 Xg for 1min and the flow-through discarded, and the above procedure was repeated once.
Add 200. mu.L of quench buffer to the first spin column, centrifuge at 1000 Xg for 1min and discard the flow through, insert bottom cap. Add 200. mu.L of quench buffer to the resin.
In a fume hood, 3. mu.L of 5mol/L sodium cyanoborohydride solution was added to the first spin column and the screw cap was capped. Mix by gentle shaking or turning upside down and incubate for 15 min.
The bottom plug is disassembled, and the screw cap is unscrewed. The first spin column was placed back into the collection tube, centrifuged at 1000 Xg for 1min and the flow-through liquid was discarded.
The screw cap was removed and the resin was washed twice with 200. mu.L of 1 Xcrosslinking buffer, after each washing 1000 Xg were centrifuged for 1 min.
The resin was washed 6 times with 150. mu.L of wash buffer, after each wash centrifugation at 1000 Xg for 1 min.
Or continuing to carry out immunoprecipitation; or the resin is stored and the process proceeds to the next step.
The resin was washed 2 times with 200. mu.L of 1 Xcrosslinking buffer, after each washing 1000 Xg were centrifuged for 1 min.
The bottom of the first spin column was removed to remove the remaining liquid and a bottom cap was inserted. Add 200. mu.L of 1 Xcrosslinking buffer, screw cap, store at 4 ℃. If long-term storage is required, sodium azide is added to a final concentration of 0.02%.
The medium in HCC1806 breast cancer cells was carefully removed.
Cells were washed twice with 1 × modified PBS, approximately 1.5ml each time.
Add 300. mu.L of ice-pre-chilled IP lysis/wash buffer to the cells, incubate for 10min on ice and mix periodically.
Incompletely lysed cells in the flask were gently scraped off with a cell scraper, and the cell lysate from the flask was transferred to a microfuge tube and centrifuged at 13000 Xg for 10min to pellet cell debris.
The supernatant was transferred to a new microcentrifuge tube and BCA protein concentration was determined to be 137.15 mg/ml.
mu.L of cell lysate, i.e., 1mg of cell lysate, was taken and 80. mu.L of control agarose resin slurry was added to the second spin column.
Centrifuge at 1000 Xg for 1min and centrifuge the column to remove storage buffer.
Add 100. mu.L of 1 Xcrosslinking buffer to the second spin column, centrifuge at 1000 Xg for 1min and discard the flow-through.
1mg of cell lysate was added to a second spin column containing resin and incubated at 4 ℃ for 1h while gently tumbling the column on a vortexer to mix.
The column was centrifuged at 1000 Xg for 1min, the second column containing the resin was discarded, and the flow-through liquid was retained ready for addition to the immobilized antibody.
The second spin column containing the resin was washed 2 times with 200. mu.L of IP lysis/wash buffer, each time 1000 Xg was used to centrifuge the column for 1min and the flow-through was discarded.
The remaining liquid at the bottom of the second centrifugation column was removed and a bottom cap was inserted.
The resulting flow-through fluid was added to 37.9. mu.L to the first spin column. And covering a screw cap, and slightly overturning the bottle on a mixed rotation instrument at room temperature for 2 hours.
Removing the bottom cover, loosening the screw cover, and placing the first centrifugal column into the collecting pipe. Centrifuge at 1000 Xg for 1min and preserve flow-through.
The screw cap was removed and the first spin column was placed in a new collection tube, 200. mu.L of IP lysis/wash buffer was added and centrifuged at 1000 Xg for 1 min.
This was followed 2 additional times with 200. mu.L of IP lysis/wash buffer, after each wash centrifugation at 1000 Xg for 1 min. The washed flow-through solution is subjected to BCA protein concentration determination, and the flow-through solution is detected for the presence or absence of protein, if any, and the washing is continued until finally the flow-through solution is free of protein.
The obtained Co-IP sample is subjected to Western Blot detection.
Comparative example
The interaction experiment between EGFR and HER2 in breast cancer cells MDA-MB-468 was performed using conventional co-Immunoprecipitation (IP) and Western Blot (WB).
Data analysis was performed using experiments with traditional co-Immunoprecipitation (IP) and Western Blot (WB) and modified IP and WB to compare the results obtained with the interaction between EGFR and HER2 in breast cancer cells MDA-MB-468 (see FIG. 1). As can be seen from FIG. 1, the method for separating and purifying proteins by using resin centrifugal column of the present invention can effectively improve the protein purity, relatively speaking, the obtained proteins have higher specificity, the occurrence of non-specific protein impurity bands in the test is avoided, and the interaction between the proteins can be accurately detected.
Compared with the prior art, the invention has the following advantages:
the method is simple and effective, has good specificity and extremely high accuracy. The combination efficiency of the protein and the protein is improved, the experiment cost is greatly saved through the regeneration and the storage of the resin, the accuracy of the experiment is improved through the reagent used for the control experiment, the efficient centrifugal column system is used, the experiment operation is further simplified, and the experiment time is shortened.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims (10)

1. A method for detecting protein-protein interactions, comprising the steps of:
step one, adding the antibody into a centrifugal column for solidification treatment;
step two, agarose matrix pretreatment of cell lysate;
step three, placing the antibody after the solidification treatment on agarose matrix pretreatment cell lysate for treatment;
step four, performing co-immunoprecipitation and elution;
and step five, performing SDS-PAGE analysis on the eluted flow-through liquid, and detecting the interaction between the target proteins.
2. The method for detecting protein-protein interaction according to claim 1, wherein the solidifying treatment in the first step comprises the steps of:
a1, pretreating the first centrifugal column, and adding an HER2 antibody, a cross-linking buffer solution and ultrapure water into the first centrifugal column;
a2, incubating by a gyrometer at room temperature, centrifuging and reserving a first centrifugal column.
3. The method for detecting protein-protein interaction according to claim 2, wherein the solute of the crosslinking buffer in step A1 is 0.01M Na3PO40.15M NaCl; the pH was 7.2.
4. The method for detecting protein-protein interaction according to claim 2, wherein the step two of pretreating the cell lysate comprises the steps of:
b1, washing the cells by a modified buffer solution, adding a precooled IP lysis/washing buffer solution, incubating on ice and centrifuging;
b2, adding agarose resin slurry into the second centrifugal column, centrifuging, adding a cross-linking buffer solution, and centrifuging again to obtain a second centrifugal column containing resin;
b3, adding the cell lysate into a second centrifugal column containing resin, and reserving flow-through liquid after incubation and centrifugation; wherein the mass-volume ratio of the cell lysate to the agarose resin slurry is 1:80 mg/muL.
5. The method of claim 4, wherein modified Duchen PBS buffer and IP lysis/washing buffer are used to lyse HCC1806 breast cancer cells in B1.
6. The method of claim 5, wherein the modified Doherty PBS has a solute of 0.008M Na3PO4、0.002M K3PO40.14M NaCl, 0.01M KCl; the pH is 7; the solutes of the IP lysis/wash buffer were 0.025M Tris, 0.15M NaCl, 0.001M EDTA, 1% NP-40, 5% glycerol; the pH was 7.4.
7. The method for detecting protein-protein interaction according to claim 4, wherein the flow-through liquid obtained from B3 is added to the first centrifugal column after A2 treatment, and the flow-through liquid is retained after centrifugation.
8. The method for detecting protein-protein interaction according to claim 1, wherein the mass of the antibody added in the first step is 10 to 75. mu.g, and the volume of the solution is adjusted to 200. mu.L.
9. The method of claim 1, wherein in step one, the antibody is covalently coupled to the amine-based activated resin.
10. The method of claim 1, wherein the step five comprises using a non-reduced lane marker loading buffer with solutes comprising 0.3M Tris-HCl, 5% SDS, 50% glycerol, and a lane marker tracer dye; the pH was 6.8.
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CN106148175A (en) * 2016-05-18 2016-11-23 北京华安麦科生物技术有限公司 A kind of method for enriched microorganism and application thereof
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龚燕华等: "《蛋白质相互作用及亚细胞定位原理与技术》", 中国协和医科大学出版社, pages: 117 - 126 *

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